Methods for generating pH/ionic concentration gradient near electrode surfaces for modulating biomolecular interactions

ABSTRACT

Device and methods for use in a biosensor comprising a multisite array of test sites, the device and methods being useful for modulating the binding interactions between a (biomolecular) probe or detection agent and an analyte of interest by modulating the pH or ionic gradient near the electrodes in such biosensor. An electrochemically active agent that is suitable for use in biological buffers for changing the pH of the biological buffers. Method for changing the pH of biological buffers using the electrochemically active agents. The methods of modulating the binding interactions provided in a biosensor, analytic methods for more accurately controlling and measuring the pH or ionic gradient near the electrodes in such biosensor, and analytic methods for more accurately measuring an analyte of interest in a biological sample.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part application of, and claimspriority under 35 U.S.C. § 120 to, U.S. patent application Ser. No.13/543,300, filed Jul. 6, 2012, and U.S. patent application Ser. No.13/834,126, filed Mar. 15, 2013, the contents of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to a biosensor device for use in diagnosticmethods for biomolecules. The invention also relates to a method andcorresponding devices and systems for detecting the presence of bubblesin an aqueous solution; and glass slides for performing life scienceexperiments, in which at least some of the processing of data measuredat the slide is performed by a computer processor located on the slideor by a processor on a peripheral component connected to a body of, orwirelessly coupled to the slide. The invention also relates to the useof electrochemical reactions, in particular redox reactions, in asolution to modulate the pH of the solution using electric current. Theinvention also relates to biological buffers and in particularelectrochemically active agents that are compatible for use inbiological buffers and are usable to facilitate pH modulation inbiological buffers. Moreover, the invention relates to a method forgenerating a pH concentration gradient near electrode surfaces formodulating biomolecular interactions in such biosensor, a method forusing integrated electronic systems for improving the accuracy,precision, and reliability in controlling a pH gradient near electrodesurfaces, methods for controlling the pH in order to modulatebiomolecular interactions in such biosensor, a diagnostic method forbiomolecules using a biosensor, and methods of improving such biosensor.

BACKGROUND INFORMATION

Recently there has been an increased interest in predictive,preventative, and particularly personalized medicine which requiresdiagnostic tests with higher fidelity, e.g., sensitivity andspecificity. Multiplexed measurement platforms, e.g., protein arrayscurrently used in research, are among the promising diagnosticstechnologies for the near future. The samples in these tests can behuman body fluids such as blood, serum, saliva, biological cells, urine,or other biomolecules but can also be consumables such as milk, babyfood, or water. Within this field there is a growing need for low-cost,multiplexed tests for biomolecules such as nucleic acids, proteins, andalso small molecules. Achieving the sensitivity and specificity neededin such tests is not without difficult challenges. Combining these testswith integrated electronics and using CMOS technology has providedsolutions to some of the challenges.

The two main limitations in a detection assay include sensitivity andcross-reactivity. Both of these factors affect the minimum detectableconcentration and therefore the diagnostic error rate. The sensitivityin such tests is generally limited by label detection accuracy,association factor of the probe-analyte pair (for example anantibody-antigen pair), and the effective density of probe molecule (forexample probe antibody) on the surface (as shown in FIG. 1). Othermolecules in the biological sample can also affect the minimumdetectable concentration by binding to the probe molecule (for examplethe primary antibody), or by physisorption of the analyte to the surfaceat the test site (as shown in FIG. 2). The detection agent (for examplea secondary antibody) may also physisorb to the surface causing anincrease in the background signal (as shown in FIG. 2). Solving thecross-reactivity and background problem can take a significant amount oftime in the assay development of a new test and increases the cost andcomplexity of the overall test. The assay is typically optimized byfinding the best reagents and conditions and also by manufacturing themost specific probe molecule (for example antibody). This results in along development time, the infeasibility of tests in some cases, and ahigher manufacturing cost. For example a typical development of an ELISAassay requires several scientists working for more than a year findingthe correct antibody as part of the assay development. Cross-reactivityof the proteins may be the source of the failure of such an effort.

A biosensor providing a multiple site testing platform was thought toprovide a solution to some of the above described limitations in assaydevelopment. US Published Patent Applications US2011/0091870 andUS2012/0115236 (the contents of which are incorporated herein byreference in their entirety) describe such biosensors having multiplesites that could be subjected to different reaction conditions tomodulate the binding of the biomolecular analyte (for example proteins)to the probe molecule. For example, the signal detected in a biosensorhaving four sites also can have several components, e.g. four. Thesefour terms may correspond to the concentration of the biomarker ofinterest, concentration of interfering analytes in the sample that bindnon-specifically to primary antibody (probe molecule) sites and preventthe biomarker to bind, concentration of interfering analytes in thesample that form a sandwich and produce wrong signal, and finally theconcentration of interfering analytes in the sample that physisorb tothe surface and produce wrong signal. Each term is also proportional toa binding efficiency factor, α_(ij), which is a function of the moleculeaffinities and other assay conditions, e.g., mass transport. Bycontrolling the condition at each site separately, different sites willhave different efficiency factors.

Accurate and precise control of the assay conditions at different sitesto generate large changes in the binding efficiency factors is importantin the performance of such biosensor as a detection system for abiomolecular analyte of interest. In US2014/0008244 (the content ofwhich is incorporated herein by reference in its entirety) suchbiosensors and such methods are described that can be readily integratedwith a CMOS, electrode array, or TFT based setup to generate largechange in binding efficiencies between test sites in a biosensor havingan array of multiple test sites. In order to accurately measure thebiomolecular analyte of interest the biosensor requires a high degree ofreliability and reproducibility. Variations in the modulation of thelocal pH due to repeated use of the biosensor and variations betweensubsequent measurements may decrease the accuracy of the determinationof the biomolecular analyte of interest by such biosensor. As such themodulation of the pH at each site of the multisite array of thebiosensor needs to be accurately controlled and variations in such pHmodulation need to be corrected. Therefore, there is a need for abiosensor in which the pH can be accurately, reliably, and reproduciblycontrolled at each of the multisite array test sites.

General methods for measuring and controlling pH are known in the art.(Durst et al., “Hydrogen-Ion Activity,” Kirk-Othmer Encyclopedia ofChemical Technology, pp. 1-15 (2009)). Active pH control of a solutionin contact with an electrode surface has potential applications inprotein-protein interactions, isoelectric focusing, electrophoresis,combinatorial pH studies of chemical and biochemical processes, DNAdenaturation and renaturation, controlling enzymatic processes, cellmanipulations, as a means for accelerating or inhibiting chemicalreactions with high spatial and temporal resolution, or in otherprocesses involving pH as a variable. For example, US2014/0008244describes a biosensor capable of modulating the pH or ionicconcentration gradient near electrodes in the biosensor in order tomodulate the binding interactions of biological samples of interest. Inanother example, US2014/0274760 (hereby incorporated by reference in itsentirety) describes an improved biosensor with increased accuracy,reliability, and reproducibility.

Attempts to control solution properties through electrochemical agentsattached to the surface have been described. Electrochemically triggeredrelease of biotin from a modified gold electrode surface via reductionand subsequent lactonization of quinone tether was demonstrated(Hodneland et al., “Biomolecular surfaces that release ligands underelectrochemical control,” J. Am. Chem. Soc. 122, pp. 4235-36 (2000)).Electrochemical control of self-assembly and release of antibodies fromthe surface into solution was achieved by reduction and oxidation ofn-decanethiol-benzoquinones (Artzy-Schnirman et al., “Artzy-Schnirman etal., Nano Lett. 2008, 8:3398-3403,” Nano Lett. 8, pp. 3398-3403 (2008)).Release of protons from a 3D layer of electroactive material wasdemonstrated by Frasconi et al. using materials composed of goldnanoparticles and thioanilines (Frasconi et al., “ElectrochemicallyStimulated pH Changes: A Route To Control Chemical Reactivity,” J. Am.Chem. Soc. 132(6), pp. 2029-36 (2010)). Electrochemical oxidation ofthioaniline groups produced protons that diffused from electrode surfaceinto the surrounding solution, thus altering its pH.

Electrochemical pH modulation in biological solutions presents asignificant challenge due to complex nature of the system. Thelimitations include: presence of buffer components that restrict pHchanges, limitations on co-solvents that can be used, presence of strongnucleophiles, such as amines and thiols, and presence of interferingelectrochemically active components, such as DNA bases, ascorbic acidand glutathione.

Quinones are one of the most widely studied classes of electrochemicallyactive molecules (See Thomas Finley, “Quinones,” Kirk-OthmerEncyclopedia of Chemical Technology, 1-35 (2005), which is incorporatedby reference in its entirety. See also, Chambers, J. Q. Chem. QuinonoidCompd. 1974, Pt. 2:737-91; Chambers, J. Q. Chem. Quinonoid Compd. 1988,2:719-57; Evans, D. H. Encycl. Electrochem. Elem. 1978, 12: 1-259).Hydroquinone/benzoquinone transformation has been used as a model systemto produce proton gradients at electrode surface (Cannan et al.,Electrochem. Communications 2002, 4:886-92). A combination ofpara-hydroquinone and anthraquinone was used for generation of acidic pHin organic solution as a first step of DNA synthesis, and organic basewas added to the solution in order to confine the acidic pH to electrodesurface (Maurer, PLOS One 2006, 1:e34). However, those systems cannot beadopted for use in biological solutions due to reactivity ofbenzoquinone (the product of hydroquinone oxidation) towardsnucleophiles that are often present in biological systems, such aspeptides, proteins and glutathione (Amaro et al., Chem Res Toxicol 1996,9(3):623-629); and further due to the insufficient solubility ofunsubstituted anthraquinone in water.

Electrochemical time of flight measurements have demonstrated that H⁺ions generated on electrodes will diffuse out (Slowinska et al., “Anelectrochemical time-of-flight technique with galvanostatic generationand potentiometric sensing,” J. Electroanal. Chem. Vol. 554-555, pp.61-69 (2003); Eisen et al., “Determination of the capacitance ofsolid-state potentiometric sensors: An electrochemical time-of-flightmethod,” Anal. Chem. 78(18), pp. 6356-63 (2006)). It has also been shownthat the open circuit potential of an electrode surface is a function ofthe ionic concentration in a solution, including the H⁺ concentration inthe solution, and therefore of the pH of the solution (Yin et al.,“Study of indium tin oxide thin film for separative extended gateISFET,” Mat. Chem. Phys. 70(1), pp. 12-16 (2001)). Similarly, the redoxreaction rates of electrochemical species are also pH dependent (Quan etal., “Voltammetry of quinones in unbuffered aqueous solution:reassessing the roles of proton transfer and hydrogen bonding in theaqueous electrochemistry of quinones,” J. Am. Chem. Soc. 129(42), pp.12847-56 (2007)). There has also been work done on improving the pHsensitivity of an electrode by incorporation of novel pH sensitivecoatings to improve the accuracy of pH sensing (Ge et al.; “pH-sensingproperties of poly(aniline) ultrathin films self-assembled on indium-tinoxide,” Anal. Chem. 79(4), pp. 1401-10 (2007)).

Many life science applications (proteomics, genomics, microfluidics,cell culture, etc.) use glass slides as a substrate for performingexperiments. Examples of glass slides include protein microarrays,lysate arrays, DNA microarrays and cell culture platforms. One use of aprotein microarray is to analyze biological substances (e.g., bloodserum) from patients with a specific disease in comparison tocorresponding substances from healthy or control subjects. Thebiological substances are applied to a microarray containing many (oftenthousands of) human proteins. Antibodies in diseased substances mayreact (bind) with certain antigens in the microarray, therebyidentifying the antigens as disease-specific biomarkers. In addition toprotein detection, other types of detection such as colorimetric,chemiluminescence and fluorescence detection are also possible withglass slides.

Often the experiments are performed under aqueous conditions, in which asubstance-of-interest is combined with water or a water-containingliquid and placed onto a slide for analysis. In many cases the presenceof bubbles (formed of air or other gases) disturbs the experiment,adversely affecting the results. One example of an adverse effect iswhen a bubble causes the test solution to dry out. This can create afalse binding event where the substance-of-interest (e.g., abiomolecular analyte) fails to bind with a molecule with which thebiomolecular analyte is supposed to interact. Another example is wherethe bubbles change the effective flow rate of the test solution and theflow rate is being measured as part of the experiment. Therefore, it isdesirable to detect bubbles and to output an indication of theirpresence, so that experiment results can be interpreted correctly.

One way to detect bubbles is to manually check each slide under amicroscope. However, microscopy is not always practical because thefield of view is typically limited to a small area of the slide, so thatchecking the entire slide is time-consuming. Additionally, the use oflight to illuminate the slide under the microscope can sometimes have adestructive effect on the substance-of-interest.

SUMMARY OF THE INVENTION

Herein are provided such methods that can be integrated with for examplea CMOS, electrode array, or TFT based biosensor to generate largechanges in binding efficiencies between test sites in the biosensorhaving an array of multiple test sites. In particular, the currentapplication provides methods to modulate the pH or ionic concentrationnear electrode surfaces of such biosensors in order to modulate thebiomolecular interactions between a probe biomolecule and a biomolecularanalyte of interest.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized probes thereon;

b) adding an electrochemically active agent, an enzyme, an enzymesubstrate, a buffer inhibitor, or a combination thereof to the aqueoussolution; and

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or a combination thereof in the aqueous solution to produceH⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ionswith the buffer inhibitor, or inhibiting the interaction between H⁺ ionsor OH⁻ ions and buffering salts with the buffer inhibitor.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized probes;

b) adding an electrochemically active agent to the aqueous solution; and

c) oxidizing or reducing the electrochemically active agent.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized probes and one or more immobilized enzymes thereon;

b) adding an enzyme substrate to the aqueous solution; and

c) enzymatically oxidizing or reducing the enzyme substrate.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising an electromagnet in anaqueous solution and a biomolecular interface layer having one or moreimmobilized probes;

b) adding one or more enzymes immobilized onto magnetic micro- ornano-particles to the aqueous solution;

c) adding an enzyme substrate to the aqueous solution; and

d) enzymatically oxidizing or reducing the enzyme substrate.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized probes;

b) adding an enzyme to the aqueous solution; and

c) reacting the enzyme at the electrode surface.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized probes;

b) adding a buffer inhibitor to the aqueous solution; and

c) inhibiting the diffusion of H⁺ ions or OH⁻ ions or the interactionbetween H⁺ ions or OH⁻ ions and buffering salts.

According to example embodiments, there is provided a biosensor for usein detecting a biomolecule analyte comprising a multisite array of testsites in which the conditions for interacting with the biomoleculeanalyte can be varied independently, each test site comprising:

a) a support in an aqueous environment;

b) one or more electrodes; and

c) a biomolecular interface layer having one or more immobilized probesand one or more immobilized enzymes.

According to example embodiments, there is provided a method fordetecting a biomolecule analyte in a biological sample, the methodcomprises:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution comprising a water-miscible organic co-solvent, e.g.,acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), andN,N-dimethyl acetamide (DMAc), to facilitate the dissolution of anelectrochemical active agent, the support comprising one or moreelectrodes or an electromagnet, and a biomolecular interface layerhaving one or more immobilized probes thereon;

b) adding in each test site an electrochemically active agent, anenzyme, an enzyme substrate, a buffer inhibitor, or a combinationthereof to the aqueous solution;

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or a combination thereof in the aqueous solution to produceH⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ionswith the buffer inhibitor, or the inhibiting the interaction between H⁺ions or OH⁻ ions and buffering salts with the buffer inhibitor;

d) adding a biological sample to each test site; and

e) detecting the biomolecule analyte in each test site,

wherein the amounts added in step b) and the reaction in step c) arevaried between test sites in a subset array of test sites in order toobtain sets of test sites in which the pH or ionic concentration nearthe electrode surfaces in the test sites varies.

Also provided are devices and methods for accurately, reliably andreproducibly controlling the pH that can be integrated with for examplea CMOS, electrode array, or TFT based biosensor having an array ofmultiple test sites. In particular, the current application providesmethods to reliably and reproducibly modulate the pH or ionicconcentration near electrode surfaces of such biosensors in order tomodulate the biomolecular interactions between a probe biomolecule and abiomolecular analyte of interest. The device described herein can beused in a biosensor in order to repeatedly determine a biomolecularanalyte of interest in a sample while maintaining a high degree ofaccuracy of the biosensor.

According to example embodiments, there is provided a device for use ina biosensor having a multisite array of test sites, the devicecomprising:

-   -   (a) a support substrate supporting one or more electrodes; and    -   (b) a biomolecular interface layer having immobilized pH        sensitive Fluorescent Protein and one or more immobilized probes        thereon.

According to example embodiments, there is provided biosensor comprisingthe device comprising:

-   -   (a) a support substrate supporting one or more electrodes; and    -   (b) a biomolecular interface layer having immobilized pH        sensitive Fluorescent Protein and one or more immobilized probes        thereon.

According to example embodiments, there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

-   -   a) providing a biosensor comprising a multisite array of test        sites in which the conditions for interacting with a biomolecule        analyte can be varied independently, the biosensor having a        device comprising a support substrate supporting one or more        electrodes, and a biomolecular interface layer having        immobilized pH sensitive Fluorescent Protein and one or more        immobilized probes thereon; and    -   b) reacting at the one or more electrodes an electrochemically        active agent in an aqueous solution to produce H⁺ ion or OH⁻        ions.

According to example embodiments, there is provided a method fordetecting a biomolecule analyte in a biological sample, the methodcomprising:

-   -   a) providing a biosensor comprising a multisite array of test        sites in which the conditions for interacting with a biomolecule        analyte can be varied independently, the biosensor having a        device comprising a support substrate supporting one or more        electrodes and a biomolecular interface layer having immobilized        pH sensitive Fluorescent Protein and one or more immobilized        probes thereon, and at each test site having an aqueous solution        comprising a dilute phosphate buffer and an electrochemically        active agent;    -   b) at each test site electrochemically reacting the        electrochemically active agent in an aqueous solution to produce        H⁺ ion or OH⁻ ions, thereby modulating and controlling the pH at        each test site;    -   c) adding a biological sample to each test site; and    -   d) detecting the biomolecule analyte in each test site,

wherein the amounts of electrochemically active agent and theelectrochemical reaction are varied between test sites in a subset arrayof test sites in order to obtain sets of test sites in which the pH orionic concentration near electrode surfaces in the test sites varies,and wherein the pH at each test site is determined by the fluorescenceintensity of the pH sensitive Fluorescent Protein.

Also provided is a system and method for detecting bubbles in an aqueoussolution. The solution is placed onto a measurement area of a slide, themeasurement area containing a plurality of electrodes configured tomeasure capacitance. According to example embodiments, the slide isconfigured to support measurements of at least one additional type ofdata in connection with an experiment involving a substance-of-interestcontained in the solution. This allows the slide to be used forconventional testing purposes in addition to bubble detection. Accordingto an example embodiment, one or more electrodes are configured for dualfunctioning so that the one or more electrodes help facilitate both theconventional testing function and the bubble detection.

According to example embodiments, the slide includes a control unit thatmeasures capacitance values associated with individual ones of theelectrodes and/or pairs of the electrodes. The control unit captures thecapacitance value(s) for subsequent processing and allows for capturingof bubble detection relevant data, obviating the need to manually checkthe slide for bubbles.

According to example embodiments, the system and method includeanalyzing measured capacitances to identify a location of a bubble inthe solution. In an example, bubbles are detected based on comparing thevalue of the capacitances to a threshold value or to other measuredcapacitances. This allows the location of the bubbles to be determinedwithout user input.

According to example embodiments, the system and method involvedisplaying bubble locations graphically, preferably as athree-dimensional graph. This allows a user to quickly determine wherebubbles are located, without having to manually interpret the measuredcapacitance values. The user can then determine, based on the bubbleindications, whether to keep or discard additional data that is beingmeasured as part of the experiment. In an example, the additional datais automatically invalidated by a computer processor in response tobubble detection, to further reduce user burden.

According to example embodiments, the system and method involve usingthe slide to adjust the pH level of the solution using at least some ofthe same electrodes that are used for measuring capacitance inconnection with bubble detection. This allows for more efficient usageof the electrodes and provides an additional level of functionality tothe slide.

According to example embodiments, the system and method include using acomputer processor to perform at least some of the processing requiredby the system and method, prior to outputting the data to an externalcomputer. The processor is powered by a small power source, on orconnectable to the slide. The processor, in combination with the powersource, allows routine processing to be performed in a power efficientmanner.

Also provided are electrochemically active compositions having quinonederivatives where the reactivity between a nucleophile and the quinonederivative is reduced as compared to the reactivity between thenucleophile and an unsubstituted quinone from which quinone derivativeis derived, and the composition is configured such that the pH of thecomposition is electrochemically modulated via the quinone derivative.The composition can be added to a solution and is suitable forelectrochemical pH modulation in biological buffers. The presentinvention also provides methods of making the quinone derivatives and/orcompositions, and uses thereof.

According to example embodiments, electrochemically active compositionsare provided as an aqueous solution and optionally further comprise oneor more additive selected from the group consisting of: an aqueousbuffer, an organic solvent, an electrolyte, a buffer salt, a bioreagent,a biomolecule, a surfactant, a preservative, a cryoprotectant, andcombinations thereof. According to example embodiments, the compositioncomprises one or more nucleophiles and a quinone derivative. The pH ofthe composition is able to be electrochemically modulated throughelectrochemically induced redox reactions of the quinone derivatives insolution.

According to example embodiments, methods for modifying unsubstitutedquinones to reduce their reactivity with nucleophiles comprisesubstituting one or more hydrogen of the unsubstituted quinone with asubstituent (R-group) to provide a quinone derivative with a reducedreactivity with nucleophiles compared to the reactivity between theunsubstituted quinone and the nucleophile.

According to example embodiments, methods for increasing the watersolubility of a quinone comprise substituting one or more R groups ofthe quinone with a polar group to provide a water soluble quinonederivative. The polar group has atoms containing lone pair electrons andis capable of forming hydrogen bonds with water.

According to example embodiments, methods for synthesizing substitutedmethyl quinone comprise the following reaction steps: (i) reacting astarting material with a hydrogen halide in the presence of acetic acidand an aldehyde; (ii) reacting a material produced by step (i) with anucleophile of structure R—X, where X is either OH, NH₂, NHR, SH, O⁻, orS⁻ and R is a substituent, (iii) reacting a material produced by step(ii) with an oxidizing agent; and (iv) reacting a material produced bystep (iii) with a reducing agent.

According to example embodiments, methods of modulating the pH of asolution comprise providing to the solution any one of the quinonederivatives described above, or a combination thereof providing anelectrical current to the solution resulting in an electrochemicalreaction that reduces or oxidizes the quinone derivative; measuring thepH of the solution; and modifying the electric current to modulate thepH of the solution.

According to example embodiments, methods for adjusting anoxidation/reduction potential of a quinone comprise substituting one ormore hydrogen with an electron withdrawing or electron donating group.

Also provided are integrated systems for using electronics to change thepH of a solution close to an electrode in a controlled fashion.Moreover, the present invention provides control methods for theintegrated system that allow for generating precisely controlled changesin the pH of a solution close to an electrode.

According to example embodiments, methods for changing the pH of asolution by electronic control comprise applying an electric source tothe solution using two or more electrodes to electrochemically generateand/or consume hydrogen ions in the solution. The generation and/orconsumption of the hydrogen ions are achieved by an electrochemicalreaction of one or more redox active species in the solution.

According to example embodiments, methods for controlling the pH of asolution using two or more electrodes comprise obtaining an open circuitpotential (OCP) of the two or more electrodes in the solution, while noelectric input is being applied between the two or more electrodes,selecting an amount of electric input based on the OCP, and providingthe selected amount of electric input to the solution between the two ormore electrodes to change the pH of the solution.

According to example embodiments, methods for monitoring the pH of asolution using a sense electrode, a reference electrode, and a workingelectrode comprise selecting a target open circuit potential (OCP) forthe solution, characterizing an OCP of the solution between thereference electrode and the sense electrode while no electric input isbeing applied to the working electrode, and iteratively performing thefollowing steps: selecting an amount of electric input to be applied tothe working electrode in order to minimize a difference between the OCPof the solution and the target OCP; and applying the amount of electricinput to the working electrode to adjust the OCP of the solution.

According to example embodiments, a device for controlling the pH of asolution comprises a controller, two or more electrodes, and a solutioncontaining one or more redox active species. The device is configured toobtain the open circuit potential (OCP) between the two or moreelectrodes in the solution to generate a OCP data and send the OCP datato the controller, and the controller is configured to iterativelyperform the following steps: select an amount of electric input based ona difference between a target OCP and the obtained OCP data, apply theamount of electric input to the solution.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Illustration of the steps of a typical and well known ELISAassay: a) Sample introduced to immobilized primary antibody on a blockedsurface and incubated, b) Sample washed, and c) labeled secondaryantibody is added. The number of labels is proportional to theconcentration of target antigen.

FIG. 2: Illustration of the undesired cross-reactivity. Molecules otherthan the antigen of interest (diamond) can bind to primary antibody orthe surface and either create incorrect signal or prevent the antigen informing a sandwich.

FIG. 3: Illustration of the multisite sensor and the components in thedetected signal. The two schematics on the bottom correspond to two ofthe sites.

FIG. 4: Illustration of the composition of a sensor test site in amultisite sensor.

FIG. 5: Schematic of the pH change on an electrode surface usingelectrochemical method.

FIG. 6: Illustration of pH change by enzymatic reactions when they arebrought close to the protein surface using magnetic micro/nanoparticles.The micro/nano cavity helps in localizing the pH change.

FIG. 7A: Cyclic voltammograms of Indium Tin Oxide (ITO) electrodes inPBS only. The region where pH change can occur is where there is oxygenevolution more than 1V in respect to Ag/AgCl reference electrode.

FIG. 7B: Cyclic voltammetric study of the oxidation of Ascorbic acidtest in ITO electrodes.

FIG. 8A: Application of 1V on the ITO-PEG surface in Phosphate buffer.Impedance changes before and after application of 1V indicates thechanges or removal of PEG from electrode.

FIG. 8B: Oxidation of ascorbic acid at 0.5V and 0.75V at ITO-PEGsurface. No change in impedance during ascorbic acid oxidation indicatesPEG layers do not undergo any change.

FIG. 9: Illustration of a substrate (glass or plastic) (1) with an arrayof electrodes (2) onto which a biomolecular interface layer (10) isapplied which include fluorescence protein (such as Green FluorescenceProtein (GFP)) spots (5), and immobilized probes (4), immobilized usinga polyethylene glycol (PEG) linker (3).

FIG. 10: shows the change in the fluorescence intensity of GFPcovalently bound to the PEG-coated ITO in response to the change insolution pH. The solution pH was adjusted by adding HCl to a dilutephosphate buffer (pH 7.4).

FIG. 11: shows the pH change at the surface of ITO working electrodegenerated via current-driven oxidation of a redox active molecule,2-methyl-1,4-dihydroquinone, in diluted phosphate buffer (pH=7.4)containing 0.1M Na₂SO₄. After 10 seconds of induction, current (50microamps) was applied for 30 second, which resulted in a drop ofsolution pH to 5.5, as was observed by a change in GFP fluorescenceintensity (FIG. 10 is used as calibration curve to assess the pHvalues). After current was turned off, the pH recovered to neutral valuewithin 50 seconds.

FIG. 12: illustrates the visual changes in the GFP spot before, duringand after pH modulation experiment. A, the profile of fluorescenceintensity across the spot is shown. B, the changes in the GFP spotfluorescence intensity are shown before (0 sec), during (40 sec), andafter (110 sec) applying a current through an electrode.

FIG. 13: shows a glass slide with an ASIC chip interfaced to transparentITO electrodes.

FIG. 14: is a block diagram of a system for bubble detection, accordingto an example embodiment of the present invention.

FIG. 15: is a top view of an example electrode array, according to anexample embodiment of the present invention.

FIG. 16 and FIG. 17: show different electrode shapes, according toexample embodiments of the present invention.

FIG. 18 and FIG. 19: show a simplified electrical model of a slide thatprovides bubble detection, according to an example embodiment of thepresent invention.

FIG. 20 to FIG. 22: are graphs showing simulated capacitance values withand without the presence of a bubble.

FIG. 23: is a graph showing actual measured capacitance values with andwithout the presence of a bubble.

FIG. 24: is a flowchart of a method for detecting bubbles, according toan example embodiment of the present invention.

FIG. 25: is a simplified schematic of a circuit for calculatingcapacitance, according to an example embodiment of the presentinvention.

FIG. 26: is a flowchart of a method for pH modulation, according to anexample embodiment of the present invention.

FIG. 27 to FIG. 29: each show a slide with data processing capability,according to an example embodiment of the present invention.

FIG. 30: shows a graphic representation of a system for electrochemicalpH generation comprising a biological buffer with an electrochemicallyactive agent dissolved in bulk solution over an electrode, where thechange in pH is confined to the vicinity of the electrode surfacethrough the buffering action of bulk solution, according to an exampleembodiment of the present invention.

FIG. 31: provides structures of hydroquinones and benzoquinones that canbe used for pH generation in biological solutions, according to anexample embodiment of the present invention.

FIG. 32: demonstrates an effect of substitution in benzoquinones on thestability of proteins according to example embodiments of the presetinvention.

FIG. 33: shows square wave voltammograms of substituted quinones inbuffered solution (vs. Ag/AgCl) according to example embodiments of thepresent invention.

FIG. 34: shows steps for the synthesis of substituted hydroquinones andbenzoquinones.

FIG. 35: illustrates an open loop waveform used to maintain the pH of asolution close to an electrode, according to an example embodiment ofthe present invention.

FIG. 36: illustrates examples of waveform shaping for pH control,according to example embodiments of the present invention.

FIG. 37: illustrates a response of the open circuit potential for a PANIcoated surface as a function of pH, where the 60 mV/pH is close to theNernstian limit, according to an example embodiment of the presentinvention.

FIG. 38: illustrates, in part A, a controlled OCP on a sense electrode(SE) by using a closed loop feedback method with a single OCPV_(TARGET), and illustrates, in part B, experimental results of acontrolled OCP on the SE by using a closed loop feedback with definedupper and lower OCP V_(TARGET) values, according to an exampleembodiment of the present invention.

FIG. 39: shows experimental results of a closed look feedback method forcontrolling the pH of the solution as represented by the open circuitpotential voltage measured by the sense electrode (SE) by applying apotential to the working electrode (WE), according to an exampleembodiment of the present invention.

FIG. 40: illustrates open loop schematics, in part A, a controlledcurrent source is used, while in part B, a controlled voltage source isused, according to an example embodiment of the present invention.

FIG. 41: illustrates a closed loop single controlled current source,according to an example embodiment of the present invention.

FIG. 42: illustrates a closed loop dual controlled current source,according to an example embodiment of the present invention.

FIG. 43: illustrates a closed loop single controlled current source witha PANI coated sense electrode, according to an example embodiment of thepresent invention.

FIG. 44: illustrates a closed loop dual controlled current source with aPANI coated sense electrode, according to an example embodiment of thepresent invention.

FIG. 45: illustrates a closed loop single controlled current source witha combined working and sense electrode, according to an exampleembodiment of the present invention.

FIG. 46: illustrates a closed loop dual controlled current source with acombined working and sense electrode, according to an example embodimentof the present invention.

FIG. 47: illustrates a closed loop single controlled current source witha combined working and sense electrode with PANI coating, according toan example embodiment of the present invention.

FIG. 48: illustrates a closed loop dual controlled current source with acombined working and sense electrode with PANI coating, according to anexample embodiment of the present invention.

FIG. 49: illustrates a closed loop single controlled potential sourcewith a PANI coated sense electrode, according to an example embodimentof the present invention.

FIG. 50: illustrates a closed loop dual controlled potential source witha PANI coated sense electrode, according to an example embodiment of thepresent invention.

FIG. 51: illustrates a closed loop single controlled potential sourcewith a combined working and sense electrode, where feedback controlledPhi1 and Phi2 switches are for WE potential input and SE measurementoutput, according to an example embodiment of the present invention.

FIG. 52: illustrates a closed loop dual controlled potential source witha combined working and sense electrode, where feedback controlled Phi1and Phi2 switches are for WE potential input and SE measurement output,according to an example embodiment of the present invention.

FIG. 53: illustrates a closed loop single controlled potential sourcewith a combined working and sense electrode with PANI coating, wherefeedback controlled Phi1 and Phi2 switches are for WE potential inputand SE measurement output, according to an example embodiment of thepresent invention.

FIG. 54: illustrates a closed loop dual controlled potential source witha combined working and sense electrode with PANI coating, where feedbackcontrolled Phi1 and Phi2 switches are for WE potential input and SEmeasurement output, according to an example embodiment of the presentinvention.

FIG. 55: illustrates a closed loop dual controlled current source withseparate working and sense electrode with PANI coating and an analogcontroller architecture with analog signal processing for closed loopfeedback control, the architecture being applicable to the controllerschematics designated in FIGS. 41-54, according to an example embodimentof the present invention.

FIG. 56: illustrates a closed loop dual controlled current source withseparate working and sense electrode with PANI coating and a digitalcontroller architecture with digital signal processing for closed loopfeedback control, the architecture being applicable to the controllerschematics designated in FIGS. 41-54, according to an example embodimentof the present invention.

FIG. 57: illustrates potential electrode configurations (routing notshown) for pH sensing, according to example embodiments of the presentinvention.

DETAILED DESCRIPTION

In order to vary the pH or ionic concentration gradient in a multisitearray of test sites in a biosensor there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized probes thereon;

b) adding an electrochemically active agent, an enzyme, an enzymesubstrate, a buffer inhibitor, or a combination thereof to the aqueoussolution; and

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or a combination thereof in the aqueous solution to produceH⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ion or OH⁻ ionswith the buffering agent or inhibiting the interaction between H⁺ ionsor OH⁻ ions and buffering salts with the buffer inhibitor.

In the above described method a local pH or ionic concentration gradientcan be obtained in the various test sites in a multisite arraybiosensor. The variation of the local pH and/or ionic concentrationgradient at the electrode, and in particular in the vicinity of the(biomolecular) probe in a biomolecular interface layer, over subsets ofthe multisite array of the biosensor, allows for modulating the bindingefficiency of the (biomolecular) probe and an analyte to be tested froma biological sample. The analyte of interest, when bound to the(biomolecular) probe, can be then detected using a detection agent, suchas for example a labeled secondary antibody. The modulation of bindingefficiencies in a subset of a multisite array provides a method for theaccurate determination of such analyte of interest.

The biosensor preferably comprises a multisite array of test sites asfor example is described in US 2011/0091870. Such multisite arraypreferably includes a number of different subarrays/subsets of testsites. Each test sites represents a site for performing an analysis of a(biomolecular) analyte from a biological sample through the detection ofthe (biomolecular) analyte using a (biomolecular) probe. The analyticalconditions in each test site in each of the subarrays/subsets may bevaried to obtain a collection of varied signals that will result inmultiple equations and multiple unknowns from which the concentration ofthe (biomolecular) analyte can be determined in order to obtain anaccurate measurement of the (biomolecular) analyte.

The multiple unknowns in the obtained varied signals each includes aterm that is proportional to a binding efficiency factor, α_(ij), andthe concentrations of the various molecules in the biological samplebinding that are detected at the test site. The multiple equations withmultiple unknowns may be represented for example as follows,

where C_(an) corresponds to the targeted biomolecular analyteconcentration and C_(j1), C_(j2), C_(j3) correspond to the totalconcentration of molecules which result in different terms in backgroundsignal, from which collection of multiple equations the concentration ofthe targeted biomolecular analyte can be determined.

The number of subarrays/subsets, as well as the number of test siteswithin each subarray/subset may be varied, as needed to obtain suchaccurate measurement of the analyte. Some of these analytical conditionsinclude parameters such as for example temperature, shear stress, andpressure. For example the temperature of the aqueous solution in whichthe biomolecular probe and analyte of interest in the biological sampleinteract can be varied using the electromagnetic heat at the test site.Another important condition for the interaction between the biomolecularprobe and the analyte of interest is the pH or ionic concentration. Themethod described herein modulates this pH or ionic concentration in thelocal environment of the biomolecular probe in order to affect thebinding efficiency in the vicinity of the biomolecular probe.

Each test site in the subarray/subset of the multiple site arraycomprises a support onto which one or more electrodes are placed andonto which solid surface the biomolecular probe(s) are immobilized orbound (as shown in FIGS. 121.3 and 121.4). This immobilization ofbiomolecular probes to a solid surface or support assists in reducingthe amount of probe needed for the analytical method and also localizesthe detection area to make accurate measurements. The biomolecularprobes are therefore attached to solid surfaces of the support and/orelectrodes such as those of silicon, glass, metal and semiconductormaterials (as shown in FIG. 4).

The biomolecular probe is attached or immobilized onto the supportand/or electrode(s) within a biomolecular interface layer (as shown inFIG. 4). The biomolecular layer includes a layer of immobilizedpolymers, preferably a silane immobilized polyethylene glycol (PEG).Surface-immobilized polyethylene glycol (PEG) can be used to preventnon-specific adsorption of biomolecular analytes onto surfaces. At leasta portion of the surface-immobilized PEG can comprise terminalfunctional groups such as N-hydroxysuccinimide (NHS) ester, maleimide,alkynes, azides, streptavidin or biotin that are capable of conjugating.The biomolecular probe may be immobilized by conjugating with thesurface-immobilized PEG. It is important that the method used to changethe pH does not impair the covalent binding of for example the PEG ontothe surface of a solid support, or the linker that conjugated thebiomolecular probe to the PEG (as shown in FIG. 5). The method ofmodulating the pH or ionic concentration as described herein can protectthese surface chemistries, while affecting a pH/ionic concentrationchange in the environment of the biomolecular probe.

A suitable biomolecular probe can be a carbohydrate, a protein, aglycoprotein, a glycoconjugate, a nucleic acid, a cell, or a ligand forwhich the analyte of interest has a specific affinity. Such probe canfor example be an antibody, an antibody fragment, a peptide, anoligonucleotide, a DNA oligonucleotide, a RNA oligonucleotide, a lipid,a lectin that binds with glycoproteins and glycolipids on the surface ofa cell, a sugar, an agonist, or antagonist. In a specific example, thebiomolecular probe is a protein antibody which interacts with an antigenthat is present for example in a biological sample, the antigen being abiomolecular analyte of interest.

In the analytical method described herein the analyte of interest in abiological sample can be for example a protein, such as an antigen orenzyme or peptide, a whole cell, components of a cell membrane, anucleic acid, such as DNA or RNA, or a DNA oligonucleotide, or a RNAoligonucleotide.

A biosensor comprising the device provided herein can be used in ananalytical method for determining a biomolecular analyte of interest ina biological sample, which can be for example a protein, such as anantigen or enzyme or peptide, a whole cell, components of a cellmembrane, a nucleic acid, such as DNA or RNA, or a DNA oligonucleotide,or a RNA oligonucleotide.

In such method a local pH or ionic concentration gradient can beobtained at various test sites in a multisite array biosensor. Thevariation of the local pH and/or ionic concentration gradient at theelectrode, and in particular in the vicinity of the (biomolecular) probein a biomolecular interface layer, over subsets of the multisite arrayof the biosensor, allows for modulating the binding efficiency of the(biomolecular) probe and an analyte to be tested from a biologicalsample. The analyte of interest, when bound to the (biomolecular) probe,can be then detected using a detection agent, such as for example alabeled secondary antibody. The modulation of binding efficiencies in asubset of a multisite array provides a method for the accuratedetermination of such analyte of interest.

The electrodes can be any electrode suitable in a biosensor for exampleindium tin oxide (ITO), gold, or silver electrodes. In a preferredembodiment the electrodes in the biosensor in the method describedherein are indium tin oxide (ITO) electrodes.

This analytical method using a biosensor for detecting a biomoleculeanalyte in a biological sample according to one embodiment is a methodwhich comprises the steps of:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecular interface layer having one or moreimmobilized detection agents thereon;

b) adding in each test site an electrochemically active agent, anenzyme, an enzyme substrate, a buffer inhibitor, or a combinationthereof to the aqueous solution;

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or combination thereof in the aqueous solution to produce H⁺ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ions withthe buffer inhibitor, or inhibiting the interaction between H⁺ ions orOH⁻ ions and buffering salts with the buffer inhibitor;

d) adding a biological sample to each test site; and

e) detecting the biomolecule analyte in each test site, wherein theamounts added in step b) and the reaction in step c) are varied betweentest sites in a subset array of test sites in order to obtain sets oftest sites in which the pH or ionic concentration near the electrodesurfaces in the test sites varies. The aqueous solution comprises awater-miscible organic co-solvent, e.g., acetonitrile, dimethylsulfoxide (DMSO), dimethyl formamide (DMF), and N,N-dimethyl acetamide(DMAc), to facilitate the dissolution of an electrochemical activeagent. The amount of water-miscible organic solvent can range from 0.1to 80% v/v, preferably from 0.1 to 10% v/v, most preferably from 1.0 to5.0% v/v in respect to water in the aqueous solution. The analyticalmethod hereby obtains in each subset of test sites a pH or ionicconcentration gradient over the test sites in the subset in the vicinityof the biomolecular probe. The binding efficiencies of the analyte andany other molecule in the biological sample is thereby differentlyaffected in each series of test sites in each subset.

In another embodiment there is provided an analytical method of usingthe device described herein in a biosensor to determine the presenceand/or concentration of a biomolecular analyte of interest in abiological sample. This analytical method comprises

-   -   a) providing a biosensor comprising a multisite array of test        sites in which the conditions for interacting with a biomolecule        analyte can be varied independently, the biosensor having a        device comprising a support substrate supporting one or more        electrodes and a biomolecular interface layer having immobilized        pH sensitive Fluorescent Protein and one or more immobilized        probes thereon, and at each test site having an aqueous solution        comprising a dilute phosphate buffer and an electrochemically        active agent;    -   b) at each test site electrochemically reacting the        electrochemically active agent in an aqueous solution to produce        H⁺ ion or OH⁻ ions, thereby modulating and controlling the pH at        each test site;    -   c) adding a biological sample to each test site; and    -   d) detecting the biomolecule analyte in each test site,        wherein the amounts of electrochemically active agent and the        electrochemical reaction are varied between test sites in a        subset array of test sites in order to obtain sets of test sites        in which the pH or ionic concentration near electrode surfaces        in the test sites varies, and wherein the pH at each test site        is determined by the fluorescence intensity of the pH sensitive        Fluorescent Protein.

The biomolecular analyte can be detected using any suitable detectionmethod. Known detection methods of such analyte include luminescence,fluorescence, colorimetric methods, electrochemical methods, impedancemeasurements, or magnetic induction measurements. In various of suchmethods the analyte binds to the immobilized biomolecular probe and adetection agent such as for example a secondary labeled probe thatspecifically binds to the analyte, bound to the immobilized probe, isintroduced. This detection agent or secondary labeled probe gives riseto a detectable signal such as for example luminescence or fluorescence(as shown in FIGS. 121.5 and 121.6).

In such analytical method the pH of the solution surrounding theimmobilize biomolecular probe has been known to influence thebinding/activity between the probe and the analyte to a great extent.Concentration of other ions on surrounding proteins can also heavilyinfluence the binding/activity. Herein are provided methods to modulatethe pH and/or ionic concentration in the vicinity of the biomolecularprobe immobilized close to a surface. The modulation of the pH nearthese solid surfaces also affect the non-specific interactions of theanalyte to other molecules than the biomolecular probe and theinteractions of other molecules in the solution of the biological samplewith the biomolecular probe or analyte. The modulation of pH or ionicconcentration however should not impair any of the surface chemistries,such as those that immobilize the biomolecular probe to its solidsupport in a test site of a multisite array in the biosensor. The methodof modulating the pH or ionic concentration as described herein canprotect these surface chemistries, while affecting a pH/ionicconcentration change in the environment of the biomolecular probe.

Surface chemistry compatibility is an important consideration thatshould be noted when the methods described herein are practiced. pHchange is caused by changes in hydrogen ion or hydroxyl ionconcentrations. A variety of chemical reactions taken place atelectrode-liquid, electrode-cross linker, cross linker-protein, andprotein-protein interfaces as shown in FIG. 5 can also become ahindrance to pH changes happening near the solid surfaces to reach theproteins on top of them. They can simply act as diffusion barriers forthe ions and hinder the pH changes around the biomolecular probes andanalytes. These methods of modulating the pH or ionic concentrationdescribed herein helps in maximizing the changes in hydrogen or hydroxylion concentration so that they can overcome any diffusion barrierimposed by the surface chemistry.

Another important aspect is the buffering capacity of the solution incontact with the solid interface. The buffering effect can be largeenough that the pH change at the interface would never reach thebiomolecular probes that are immobilized away from it. The distance canvary based on the biomolecular interface layer deposited on top of thesolid interface. Such biomolecular interface layer may have a thicknessof 300 nm or less, preferable between 1-150 nm, even more preferablybetween 5-100 nm. As such the distance between the solid interface andthe biomolecular probe within the biomolecular interface layer can rangebetween 0.1-300 nm. Use of buffer inhibitors in the solution or on thesurface that extend the pH change on the electrode interface to reachthe interacting probe-analyte pair may contribute to modulating the pHor ionic concentration in the vicinity of the biomolecular probe.

Following are examples of methods for modulating the pH/ionicconcentration at the solid-liquid interfaces. These include: 1) theelectrochemical generation of ions at electrode surfaces by adding anelectrochemically active species to the solution which generates ions ofinterest (e.g., H⁺, Mg⁺, OH⁻) upon electrochemical oxidation/reduction;2) bringing enzymes close to the site of interest, which release suchions of interest from an enzyme substrate that is reacted with theenzyme; 3) the introduction of buffer inhibitors, for example, by mixingpolymers that selectively reduce the diffusion rate of ions in thesolution (e.g., phosphate). U.S. Pat. No. 7,948,015 describes the use ofsuch inhibitors for applications in which measuring small local pHchanges is of interest (e.g., in DNA sequencing). However in the methodsof locally modulating the pH similar inhibitors can be used in order toextend the local pH changes further away from the electrode-liquidinterface; and 4) the redistribution of preexisting ions near theelectrode surface due to electrostatic forces.

In one embodiment of a method for modulating the pH or ionicconcentration in a biosensor as described herein an electrochemicallyactive agent is added to the aqueous solution at a test site in amultisite array, wherein the test site has a biomolecular interfacelayer comprising a biomolecular probe or detection agent and oxidizingor reducing the electrochemically active agent. The electrochemicallyactive agent may be added at a concentration of 1 nM to 100 mM,preferably at a concentration between 10 nM and 10 mM, more preferablyat a concentration of 100 nM and 5 mM. The electrochemically activeagent may be electro-oxidized or electro-reduced at an electrodepotential in the range of −2V to +2V (vs. Ag/AgCl reference electrode).Preferably the electrode potential is in the range of −1V to +1V, evenmore preferably the electrode potential is in the range of −0.5V to+0.5V. The voltage required to drive the redox reaction can be used as areal time feedback method to monitor pH that is produced at theelectrode surface.

The device provided herein and used in a biosensor comprises such arrayof multiple test sites in solution in order to modulate the pH at eachtest site and to determine the presence and concentration of abiomolecular analyte of interest in a biological sample. In such use thedevice is in contact with an aqueous solution comprising a phosphatebuffer, preferably a diluted phosphate buffer which preferably has aconcentration of 0.1 mM to 100 mM. In a preferred embodiment the pH ofthe diluted phosphate buffer is between 5 and 8, preferably between 7and 8, and more preferably between 7 and 7.5.

Modulation of the pH or ionic concentration on a device in a biosensordescribed herein by electrochemical reaction at the one or moreelectrode may be carried out in a galvanostatic mode or potentiostaticmode. In addition, any type of electrical pulse may be applied on theelectrodes of the device in the method for modulating the pH. Such pulsemay be in the form of an annealing pulse and may vary by pulsefrequency, pulse width, and pulse shape. In an annealing pulse asufficient voltage is applied to change the pH to such that non-coventlybound molecules from the biological sample are removed from the devicein the biosensor. Such annealing pulse eliminates or reduces the needfor washing the substrate following first contact with a sample in orderto remove non-covalently bound material. Another advantage is that theannealing pulse may be more efficient to remove such non-covalentlybound material from the device than a simple washing. A preferred pulsewidth for modulating the pH is in the range of 1 nanosecond to 60minutes.

The aqueous solution may further comprise one or more additionalelectrolytes, such as for example sodium sulfate, or any other suitablestrong electrolyte. Preferably, the additional electrolyte is selectedfrom sodium sulfate, sodium or potassium chloride, sodium or potassiumbromide, sodium or potassium iodide, sodium or potassium perchlorate,sodium or potassium nitrate, tetraalkylammonium bromide andtetraalkylammonium iodide. Buffer-inhibitors may also be used in theaqueous solution. Suitable buffer inhibitors may be selected frompoly(allylamine hydrochloride), poly(diallyldimethyl ammonium chloride),poly(vinylpyrrolidone), poly(ethyleneimine), poly(vinylamine),poly(4-vinylpyridine) and tris(2-carboxyethyl)phosphine hydrochloride.When used in a method to modulate the pH such as described in co-pendingU.S. patent application Ser. No. 13/543,300 the aqueous solutionpreferably also comprises a water-miscible organic co-solvent selectedfrom the groups consisting of acetonitrile, dimethyl sulfoxide (DMSO),dimethyl formamide (DMF), N,N-dimethyl acetamide (DMAc), and mixturesthereof.

Suitable electrochemically active agents include dopamine hydrochloride,ascorbic acid, phenol and derivatives, benzoquinones and derivatives,for example, 2,5-dihydroxy-1,4-benzoquinone,2,3,5,6-tetrahydroxy-1,4-benzoquinone and2,6-dichloroquinone-4-chloroimide; naphthoquinones and derivatives, forexample, hydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone,and potassium 1,4-naphthoquinone-2-sulfonate; and 9,10-anthraquinone andderivatives, for example, sodium anthraquinone-2-carboxylate, potassium9,10-anthraquinone-2,6-disulfonate. Preferably the concentration of theelectrochemically active agent in the aqueous solution is from 1 nM to100 mM.

In another embodiment of a method for modulating the pH or ionicconcentration in a biosensor, an enzyme is immobilized in a biomolecularinterface layer also having one or more immobilized biomolecularprobers. An enzyme substrate is then added to the aqueous solution at atest site in a multisite array, wherein the test site has thebiomolecular interface layer and enzymatically oxidizing the enzymesubstrate.

In another embodiment is provided a method of modulating the pH usingthe device in a biosensor. The method of modulating the pH or ionicconcentration in a biosensor comprises:

-   -   a) providing a biosensor including one or more devices as        described herein comprising a multisite array of test sites in        which the conditions for interacting with a biomolecule analyte        can be varied independently; and    -   b) reacting at the one or more electrodes an electrochemically        active agent in an aqueous solution to produce H⁺ ion or OH⁻        ions.        In the method the concentration of the electrochemically active        agent in the aqueous solution is preferably from 1 nM to 100 mM.

In another embodiment of a method for modulating the pH or ionicconcentration, the method comprises:

a) providing a biosensor comprising an electromagnet in an aqueoussolution and a biomolecular interface layer having one or moreimmobilized detection agents;

b) adding one or more enzymes immobilized onto magnetic micro- ornano-particles to the aqueous solution;

c) adding an enzyme substrate to the aqueous solution; and

d) enzymatically oxidizing the enzyme substrate.

Suitable enzymes for immobilization in the biomolecular interface layeror onto the magnetic micro- or nano-particles include for exampleoxidases, ureases, or dehydrogenases. Such immobilized oxidase is forexample a glucose oxidase and the enzyme substrate is glucose. Theamounts of immobilized enzyme and enzyme substrate added can be variedin the different test sites in each of the subsets of the multisitearray so as to provide a pH or ionic concentration gradient in the suchsubset of the multisite array.

Alternatively the enzyme is not immobilized onto a solid surface such asin the above methods being immobilized into a biomolecular interfacelayer or onto a magnetic micro- or nano-particle but is added to theaqueous solution in the test sites of subsets of a multisite array.Through electrolysis the enzyme undergoes a redox reaction at theelectrode surface and perturbs the local pH.

In each of these embodiments the pH or ionic concentration can befurther modulated by adding a buffer inhibitor to the aqueous solution.Such addition of a buffer inhibitor either assists in diffusing theproduced ions of interest to the location of the biomolecular probe ordetection agent or inhibits the interaction of such produced ions withbuffering salts. Alternatively, in the method of modulating the pH orionic concentration in a biosensor as described herein, the bufferinhibitor is added to the aqueous solution of the test site of subsetsof a multisite array in the absence of an electrochemical active agentor immobilized enzyme. In such embodiment the buffer inhibitor is addedto the aqueous solution, and facilitates the diffusion of H⁺ ions or OH⁻ions that are produced at the electrodes in the test site or inhibitsthe interaction between H⁺ ions or OH⁻ ions and buffering salts.

Suitable buffer inhibitors include soluble polymers selected frompoly(allylamine hydrochloride), poly(diallyldimethyl ammonium chloride),poly(vinylpyrrolidone), poly(ethyleneimine), poly(vinylamine),poly(4-vinylpyridine) and tris(2-carboxyethyl)phosphine hydrochloride.The amounts of buffer inhibitor added can be varied in the differenttest sites in each of the subsets of the multisite array so as toprovide a pH or ionic concentration gradient in the such subset of themultisite array.

When methods for modulating the pH in a biosensor having a multisitearray of test sites are used in a biosensor the accuracy, reliabilityand reproducibility of the modulation of the pH at each test site isimportant. However the modulation of the pH at each test site may varybetween subsequent uses. In order to accurately determine the amount ofa biomolecular analyte of interest in a sample using the biosensor andmethod described above the pH at each test site needs to be accuratelymodulated or controlled. The device provided herein allows for accuratedetermination and control of the pH at each test site in such biosensor,the device comprising:

-   -   (a) a support substrate supporting one or more electrodes; and    -   (b) a biomolecular interface layer having immobilized pH        sensitive Fluorescent Protein and one or more immobilized probes        thereon.        The support substrate in the device described herein is        preferably a glass or plastic substrate but also be any other        non-glass substrate.

The immobilized pH sensitive fluorescent protein allows for sensing thepH at an electrode once the electrode (working electrode) causesmodulation of the pH at a particular test site such as a test site in amultisite array. The fluorescence intensity of the fluorescent proteinchanges due to modulation of the pH. The change in fluorescenceintensity of the fluorescent protein is proportional to the change inthe pH (there is a linear relationship between the pH and thefluorescence intensity). Therefore, as is also shown in FIG. 10, the pHvalue at each location at any time when the biosensor is in use can bereadily obtained by correlating the fluorescence intensity of thefluorescent protein with the pH. An accurate calibration of thecorrelation between pH and fluorescence intensity may be carried outbefore or during use of the biosensor. When the calibration is carriedout during use of the biosensor one or more test sites within amultisite array may be dedicated to calibration of the fluorescenceintensity to pH correlation. When the pH is no longer modulated at suchtest site by the electrode (working electrode) the fluorescenceintensity of the immobilized fluorescent protein reverts back to itsintensity before a current was applied through the electrode.

Preferably, the immobilized fluorescent protein is selected from animmobilized green fluorescent protein, an immobilized yellow fluorescentprotein, and an immobilized cyan fluorescent protein. More preferably,the immobilized fluorescent protein is immobilized Green FluorescentProtein (GFP). In an alternative embodiment immobilized pH sensitivedyes may be used on the support substrate instead of an immobilized pHsensitive fluorescent protein. In another alternative embodimentimmobilized pH sensitive binding proteins may be used on the substrateinstead of an immobilized pH sensitive fluorescent protein. In amultisite array of test sites in a biosensor the immobilized fluorescentprotein covers on the substrate an area that is also covered by anelectrode and an area that is not covered with an electrode. Theelectrode covered by the immobilized fluorescent protein is either aworking electrode or a counter electrode. Preferably, the immobilizedfluorescent protein is applied onto the substrate as distinct spots,wherein each spot overlaps with only one test site and an area notcovered by an electrode as shown in FIG. 12A. The presence offluorescent protein on an area that is not covered by an electrodeallows for the determination, within the biosensor, of fluorescenceintensity when the pH is not modulated by the electrode. Thisfluorescence intensity can be used as a standard and control indetermining whether, after ceasing modulation of the pH by an electrodethe fluorescence intensity will revert back to its original intensity.Accordingly, in a method for detecting a biomolecular analyte in abiological using the device, the fluorescent protein not located on ornear an electrode can be used as an internal reference for signalnormalization.

The device includes one or more counter electrodes and one or moreworking electrodes. In the device one or more electrodes can be arrangedin a multisite array, each site of the multisite array comprising aworking electrode and/or counter electrode. The electrodes can be anyelectrode suitable in a biosensor for example indium tin oxide (ITO),gold, or silver electrodes. In a preferred embodiment the electrodes inthe device are indium tin oxide (ITO) electrodes. In an alternativeembodiment the working electrode is an indium tin oxide electrode andthe counter electrode(s) is selected from an indium oxide electrode, agold electrode, a platinum electrode, a silver electrode, and a carbonelectrode.

The electrodes in the device may be used either for modulating the pH oras sensing electrodes or both. In the device or biosensor using thedevice, the one or more electrodes are connected to an electronic boardvia pogo-pins, a chip on foil via z-axis adhesive, or a chip on thesubstrate. The electronic board or chip are powered by a printedbattery, a small battery bound to the substrate, a magnetically coupledpower transfer using coils on the substrate, or a rf-coupled powertransfer using coils on the substrate.

The following description is an illustration of a specific embodimentwhich may be modified within the scope of the description as would beunderstood from the prevailing knowledge. FIG. 9, shows a side view of apart of the device which includes a substrate (1) for example glass orplastic. One or more electrodes (2) are covered onto the substrate (1)which is also covered with a biomolecular interface layer (10). Thebiomolecular interface layer (10) comprises immobilized PEG (3),immobilized probe (4) and immobilized pH sensitive fluorescent proteinin the form of Green Fluorescent Protein spots (5). The GFP spots (5)overlap with an electrode (2) and an area that is not covered by anelectrode. The electrodes (2) and the GFP spots (5) are arranged in amultisite array so as to provide multiple test sites on the device.

The location of luminescence signals generated luminescent molecules canbe controlled by directly controlling the location of the luminescentmolecules themselves. This includes for example immobilizing theluminescent molecule. However, by incorporating the ability to controlthe pH of a solution near an electrode with pH sensitive luminescentmolecules the location of luminescence signals generated by freefloating luminescent molecules can also be controlled.

Example embodiments of the present invention relate to the detection ofbubbles in glass slides, on which slides an aqueous solution is placedfor analysis. However, the example embodiments may also be appliedtowards other applications in which it is desirable to detect thepresence of bubbles. In particular, although the capacitance baseddetection techniques are described herein in connection with thecapacitive properties of water, these techniques may also be applied toother liquids for which the capacitive properties are known.

FIG. 14 shows an example system 100 for detecting bubbles according toan example embodiment of the present invention. In the example shown inFIG. 14, the system 100 includes a slide 30 that includes an area 10 inwhich a test solution containing a substance-of-interest is placed foranalysis, a control unit 12 and a power source 14. The slide 30 can beformed of any electrically insulating material. For example, glass wouldtypically be used for this purpose and to serve as a substrate, on topof which the area 10, control unit 12 and power source 14 are formed.The glass can be formed, for example, of silicon dioxide (SiO₂),possibly with additives. Alternatively, other types of silicate glassesmay be used.

The area 10 includes an array of electrodes used for bubble detection.In an example embodiment, at least some of the electrodes in the area 10are used for adjusting (also referred to herein as modulating) a pHlevel of the test solution. These pH modulating electrodes can bededicated exclusively to adjusting the pH level or, alternatively,switched between pH modulating and bubble detecting modes of operation,as will subsequently be explained. (For example, U.S. patent applicationSer. No. 13/543,300, mentioned earlier, describes the use of electrodesfor pH modulation in a biosensor, which modulation can be performedusing the electrodes discussed herein.)

FIG. 15 shows a top view of an example electrode array, in which a setof column electrodes X01 to X12 are arranged at regularly spaceddistances from each other. A set of row electrodes Y01 to Y09 are alsoarranged at regularly spaced distances and are separated from the columnelectrodes X01 to X12, e.g., by an intervening layer of glass. Eachelectrode includes one or more contact pads 31, 32 for use in bubbledetection and/or pH modulation. The shape of the pads is variable and,in an example embodiment, is substantially square. FIG. 16 shows aclose-up view of example square-shaped pads. FIG. 17 shows analternative embodiment in which the pads form an interdigitatedstructure, and are therefore frame-shaped.

In the example illustrated in FIG. 14, the control unit 12 iselectrically connected to the electrode array 10 and controls the array10 to perform bubble detection and pH modulation. The control unit 12can be, for example, a microprocessor or an application specificintegrated circuit (ASIC). In an example embodiment, the control unit 12is located on an electronic circuit board that is detachably connectedto the slide 30, e.g., using pogo pins. The control unit 12 can belocated within a packaged chip bonded directly to a rigid glasssubstrate, e.g., using a chip-on-glass process. In an alternativeexample embodiment, the slide 30 is formed of a flexible foil-typesubstrate and the control unit 12 is glued to the slide 30 using az-axis adhesive to form a chip-on-foil, in a manner similar to how chipsare bonded in certain liquid crystal displays. The control unit 12 caninclude, for example, a non-transitory computer readable storage mediumcontaining program code that implements the example bubble detection andpH modulation techniques described herein. In addition to bubbledetection, the control unit 12 can control the electrodes to performother types of sensing or to control other sensing structures, as isknown in the art of biosensors.

In an example embodiment, the control unit 12 transmits control signalsthat cause input pulses to be applied at specified electrodes.Capacitance values can be measured at the control unit 12 based on theresponses of the electrodes to the input pulses. The measurement ofcapacitance is known in the art of touch screen displays, which utilizemeasurements of self-capacitance (e.g., a single electrode) or mutualcapacitance (e.g., between two electrodes). To support bubble detection,the control unit 12 has a capacitance detection range that is greaterthan that of typical control units that measure capacitance in lifescience experiments. Control signals can also be used to apply inputpulses for pH modulation. Control signals for pH modulation can beinitiated by the control unit 12, e.g., in accordance with a predefinedprogram sequence designed for pH modulation. Alternatively, the controlsignals for pH modulation is initiated externally, e.g., in response toa command from a data processing unit 50. In an example, the controlunit 12 includes hardware and/or software components that performpreliminary signal processing on the measured capacitance values,including converting the measurements from analog to digital formatand/or filtering the measurements. In an example, the processedmeasurements are then output as raw data to the data acquisition unit40.

The power source 14 provides power to the control unit 12 and to theelectrode array 10. For example, in an example embodiment, the powersource 14 is a battery such as a coin-cell or a printed battery. In oneexample embodiment, the slide 30 is designed for one-time use and isdisposable, the battery therefore being provided with a small energycapacity, e.g., sufficient for a single measurement, and the battery canbe permanently attached to the slide, e.g., bonded or glued to the glasssurface. In an example embodiment where the slide 30 is reusable, thebattery can be rechargeable or user replaceable. Other forms of electricpower delivery may alternatively be used. In one example embodiment,electrical power is delivered wirelessly through magnetic couplingbetween an external power supply (e.g., the data acquisition unit 40)and one or more resonant coils in the slide. As an alternative tomagnetic coupling, but also using wireless power transfer, the externalpower supply may couple to the resonant coil using radio-frequency (RF)signals. In yet another example embodiment, the slide 30 receives powerthrough a wired connection to the data acquisition unit 40.

In an example, the data acquisition unit 40 is a device thatcommunicates with the slide 30 to receive the measured capacitancevalues from the control unit 12, in the form of raw data. For example,in an example, the data acquisition unit 40 includes a wiredcommunication interface 20 to a corresponding interface in the slide 30.In one example embodiment, the raw data is output from the control unit12 in parallel. For example, in an example embodiment, the control unit12 includes a plurality of output channels, with data from a single rowor a single column being output on a corresponding channel. In thisembodiment, the interface 20, for example, converts the parallel datainto a format suitable for transmission to the data processing unit 50.The conversion may involve parallel-to-serial conversion using aUniversal Asynchronous Receiver/Transmitter (UART) or other conventionaldata conversion apparatus. In an alternative embodiment, the interface20 communicates wirelessly with the slide 30, e.g., using RF signals.

In an example embodiment, the data processing unit 50 receives the rawdata from an output interface 22 of the data acquisition unit 40, e.g.,from a transmitter portion of the UART. The output interface 22 can be awired, serial interface such as a Universal Serial Bus (USB) interface.Alternatively, the output interface 22 can be wireless, e.g., aBluetooth or WiFi interface. In an example, the interface is a Bluetoothlow energy (LE) interface. The data processing unit 50 can be, forexample, a general purpose computer in the form of a desktop, a laptopor tablet, and includes, for example, a processor and a memory storinginstructions for further processing of the raw data. For example, in anexample embodiment, the further processing includes normalizing the rawdata to a predefined scale and using the normalized data to generateoutput images, such as two or three-dimensional graphs, for display atthe display device 60. Where the data processing unit 50 is a laptop ortablet, the display device 60 can be integrated into a housing of thedata processing unit 50 as a single unit. The display device 60 mayalternatively be externally connected, e.g., where the data processingunit 50 is a desktop. The output images may be combined to form a videothat shows changes in the data over time. In one embodiment, the outputimages, which represent the measured capacitance values, are displayedtogether with additional output images corresponding to other measureddata. For example, the output images and the additional output imagesmay be displayed in different portions of the same display screen oroverlaid (superimposed) on the same portion of the display screen.

In an example embodiment, the data processing unit 50 is also configuredto issue commands to the control unit 12 for pH modulation. The commandsmay be automatically generated, e.g., when a processor of the dataprocessing unit 50 determines that the pH level of the test solutionshould be adjusted. Alternatively or additionally, the commands may beuser-initiated.

According to an example embodiment, the slide 30 may include a layeredstructure in which one or more electrode layers are located on top of aglass substrate. The layered structure can be formed, for example, usinga lamination technique in which two or more layers are formed separatelyand then laminated together, e.g., using adhesive or bonding.Alternatively, the layered structure can be monolithically formed as asingle unit, using techniques known in the art of semiconductor devicefabrication. The layered structure may include one or more passivatinglayers formed, e.g., of SiO₂ (also referred to as oxide). However, itwill be understood that the composition and size of passivating layerscan vary, e.g., from an atomic layer of SiO₂ to several micrometers ofSiO₂, and formed using various techniques such as low pressure chemicalvapor deposition (LPCVD) or plasma-enhanced chemical vapor deposition(PECVD). Silicon nitride (Si₃N₄) is another example passivatingmaterial. Where the layered structure is formed using lamination, thepassivating layer can be formed as a thin film that is laminated.

The capacitance based bubble detection principles used in the exampleembodiments of the present invention will now be described withreference to FIG. 18 to FIG. 23. FIG. 18 shows a simplified electricalmodel of a slide including electrodes (ITOs 81 to 84) in an SiO₂ layer73. The ITOs 81 and 82 represent electrode pads in a first layer, e.g.,row electrode pads. The ITOs 83 and 84 represent electrode pads in asecond layer beneath the first layer, e.g., column electrode pads. TheITOs 81 to 84 are formed above a glass substrate 79, with an optionalITO layer 89 that serves as a bottommost, passivating layer. Forsimplicity, the portion of the electrode array used for pH modulation incertain example embodiments is not shown. In this simplified model, thecapacitance of the test solution is assumed to be equivalent to thecapacitance of water (CW) since the test solution is, in practice,mostly water. When there is no bubble over the electrodes, the solutionis in contact with the SiO₂ layer 73 and contributes to a seriescapacitance between ITOs 81 and 82. The SiO₂ layer 73 also contributesto the series capacitance, as represented by two capacitances CSiO₂.

The pH modulating portion of the electrode array has been omitted forthe sake of simplicity. One way to perform pH modulation is to separatethe pads of adjacent electrodes so as to form channels that collect thetest solution. The channels allow the test solution to come into contactwith the electrodes, so that the pH level of the solution can beadjusted by sending a current between the adjacent electrodes. Accordingto an example embodiment, the electrodes may be formed of any suitableconductive material, but are preferably indium tin oxide (ITO) becauseITO is transparent and relatively colorless, making it suitable forexperiments that involve optical measurements. This allows the entiremeasurement area 10 to be transparent. An oxide layer may be used as apassivating layer to cover the electrodes, similar to how the SiO₂ layer73 covers the electrodes in FIG. 18 and FIG. 19. In fact, the same oxidelayer may be used over the electrodes in both the bubble detecting andthe pH modulation portions of the electrode array. Where the pHmodulation is implemented using channels, in an example embodiment, theoxide layer does not completely fill the channels, but instead a lateralportion of the electrodes is left exposed to allow for contact with thetest solution.

FIG. 19 shows additional details regarding the electrical model of FIG.18 according to an example embodiment. In FIG. 19, the seriescapacitances are collectively represented as a mutual capacitance CL1,L1between ITOs 81 and 82. Additionally, there exists a mutual capacitancebetween ITOs 81 and 83. In an example embodiment, bubbles are detectedas a change in capacitance (e.g., in either of these mutual capacitancesor in a self-capacitance) that results when the test solution isdisplaced by a bubble, which is typically formed of air. Since air has amuch lower capacitance (the dielectric strength of air is approximatelyeighty times less than water), it is possible to detect a drop incapacitance associated with the presence of a bubble. This detectionassumes that there is no electric field on top of the bubble which wouldminimally interfere with capacitance measurements. It also assumes thatthe size of the bubble is comparable to the pad, such that most of thesurface of the pad is covered by the bubble, i.e., that no or almost notest solution is in contact with the pad. In practice, electromagneticinterference may create electric fields. Interference can be avoidedthrough setup of an appropriate, low interference testing environment.As for bubble size, in practice the size will vary dramatically and itis unavoidable that sometimes a bubble will be smaller than the pad. Tominimize the occurrence of bubbles that are smaller, each pad may besized as small as possible while balancing performance parameters suchas power consumption and maintaining inter-operative compatibility withthe control unit 12. In one embodiment the pads are less than 2millimeters wide, preferably 1 millimeter or less. This is substantiallysmaller than the size of electrodes typically used for conventionaltouch-screen applications or conventional bio-sensor applications.

FIG. 20 is a simulated graph of self-capacitance along the x direction(from the top surface of the slide towards the glass substrate) whenwater covers a pad, in an example embodiment. The graph of FIG. 20 wasgenerated based on a square pad of size 1 mm×1 mm. As shown, thecapacitance decreases continuously from a value of approximately 2×10⁻¹¹Farads at 10⁻⁶ meters down to approximately 7×10⁻¹⁴ Farads at 10⁻³meters.

FIG. 21 is a simulated graph of self-capacitance along the x directionwhen a bubble having a diameter of 1 millimeter is present on a 1 mm×1mm pad, in an example embodiment. As shown, the capacitance values aresubstantially smaller than the corresponding capacitance values fromFIG. 20 between 10⁻⁶ and 10⁻³ meters. Specifically, the capacitancestarts at approximately 7.6×10⁻¹⁴ Farads and drops beginning around3×10⁻⁵ meters to approximately 5×10⁻¹⁴ Farads. Therefore, thecapacitance with water is several orders of magnitude greater for themajority of points between 10⁻⁶ and 10⁻³ meters. Accordingly, one way todetect bubbles, according to an example embodiment of the presentinvention, is based on an evaluation of the value or the magnitude of acapacitance at any given pad, e.g., by comparing the value or magnitudeto a predefined threshold value. A bubble would then correspond to acapacitance that is less than the threshold. FIG. 20 and FIG. 21 areprovided to illustrate basic electromagnetic principles by which exampleembodiments of the present invention detect bubbles based on changes incapacitance, and are not to be construed as restricting the range ofcapacitance detection techniques that may be applicable to a system ormethod of the present invention.

Another way to detect bubbles is based on the percentage change incapacitance from water to a bubble. The simulated graph in FIG. 22,whose values were calculated using the values from FIG. 20 and FIG. 21,shows this difference in an example embodiment. In FIG. 22, thepercentage change is initially small, but starts to increase at around10⁻⁵ meters. Therefore, detection may be based on the percentage changeif the pads are suitably located, e.g., at a distance of 10⁻⁵ meters ormore from the top surface of the slide.

In addition or as an alternative to evaluating the capacitance on anindividual basis (e.g., for each pad when evaluating self-capacitance orfor a pair of pads when evaluating mutual-capacitance), detection can bebased on a comparison of capacitance values associated with a pluralityof pads, according to an example embodiment. For example, according toan example embodiment, capacitance values from a group of neighboringpads are compared to determine whether any of the capacitance values isunusually small relative to the other capacitance values. Thiscomparison is advantageous because it does not require the use of athreshold value, which may need to be adjusted based on the design ofthe slide, e.g., parameters such as pad size, shape or location.

FIG. 23 is a graph showing actual test results from a prototype slide ofan example embodiment. The graph shows mutual capacitance values betweena column electrode and a row electrode. A bubble was manually introducedat the intersection of these electrodes, beginning at around 50 seconds.The bubble was then removed and another bubble introduced at around 75seconds. This process was repeated again, with another bubble at around90 seconds. Each time a bubble was introduced, the capacitance droppedsubstantially.

Control units exist for acquiring capacitance measurements in connectionwith touch-screen applications. However, these control units aregenerally unsuitable for use with the bubble detection according to theexample embodiments of the present invention. These conventional controlunits are unsuitable because they tend to have a narrower detectionrange and lower sensitivity than what is required for the exampleembodiments. In contrast, bubble detection according to the exampleembodiments requires the ability to capture large capacitance swings(e.g., a 400 pF change in going from water to bubble) in addition to ahigh resolution in order to capture the low capacitance valuesassociated with bubbles. A typical capacitance value range for when abubble exists could be between 20 fF to 40 μF. The large range is due tothe fact that many choices are available as to the size of theelectrodes and the thickness of the passivation layer on top of theelectrodes (e.g., SiO₂, TiO₂, nitride, or no passivation layer at all).

FIG. 24 is a flowchart of a method 200 for detecting bubbles accordingto an example embodiment of the present invention. According to anexample embodiment, the method 200 is performed using the system 100.

At step 210, a capacitance (self or mutual) is measured at an electrodeor between two electrodes to measure a capacitance value and the valueis output as raw data. For example, in an example embodiment, themeasurement is performed by outputting a control signal from the controlunit 12, which control signal results in the application of an inputpulse to an electrode being measured. The control unit 12 senses theelectrical response of the electrode, e.g., by measuring a voltage or acurrent across the electrode, or between the electrode and anotherelectrode, and calculates the capacitance as a function of thisresponse. The calculation of self and mutual capacitances is known inthe art of touch screen displays. Each measured capacitance can beoutput as a raw data value to the data processing unit 50 using the dataacquisition unit 40.

FIG. 25 is a simplified schematic of a circuit 201 for measuringcapacitance according to an example embodiment of the present invention,and is provided in support of the method 200. The circuit 201 includes adecoder 220, which may be included in the control unit 12 of FIG. 14.The decoder 220 is connected via a plurality of driving lines 203, overwhich the decoder 220 sends signals to activate switches 240. Theswitches 240 may be implemented as thin-film transistors (TFTs) and areswitched to connect to respective electrodes 242 that form the electrodearray. The switches 240 are controlled by the decoder 220 to perform thecapacitance measurements, e.g., by driving a specific line 203simultaneously with an adjacent line 203. The switches 240 are furtherconnected to sensing lines 205, which in this example, form the columnsof the array. Each sensing line 205 is connected to a respectiveamplifier 230. The amplifiers 230 are in a negative feedbackconfiguration with a sensing line 205 and a capacitor 99 being connectedto a negative amplifier input, which is also connected to ground via acurrent source 235. A positive input of each amplifier 230 is connectedto reference voltage 233, which may also be ground. The voltages on thesensing lines 205 are influenced by the capacitances at the electrodes,which capacitances depend on whether a bubble is present. Thus, thevoltage outputs of the amplifiers 230 correspond to measuredcapacitances.

Returning to FIG. 24, at step 212, the measured capacitance is comparedto a threshold value. As mentioned above, the threshold value may varydepending on factors such as pad size, shape or location. However, giventhe design specifications of the slide, and in view of the abovediscussion on the bubble detection principles, one of ordinary skill inthe art would be able to compute a suitable threshold value.

Alternatively or additionally, at step 214 the measured capacitance iscompared to other measured capacitances, e.g., from a group ofneighboring electrodes or the entire set of electrodes in the array, todetect unusually low capacitances.

At step 216, the results of the comparisons are evaluated at the dataprocessing unit 60 to determine, based on the bubble detectionprinciples described earlier, whether any bubbles exist, and if so,where the bubbles are located.

At step 218, the results are output for display at the display device60. According to an example embodiment, raw data values are displayed inthe form of a two-dimensional table. Each table entry corresponds to ameasured capacitance value obtained from a corresponding electrode pad.The raw data may be displayed as a three-dimensional graph, e.g., a 3-Dmesh where the x and y values correspond to electrode locations and thez values correspond to measured capacitance values. To facilitate visualrecognition, in an example embodiment, the graph is color coded, e.g.,using a gradient scheme, e.g., a gray scale scheme or a heat map inwhich the color gradually changes until a bubble location is reached, atwhich point the color is changed to a color that contrasts the color(s)of non-bubble locations. In another embodiment, color coding is used toshow bubble locations on a two-dimensional graph in which thecapacitance values are represented using changes in color. Alternativelyor additionally to the display of raw data, the data processing unit 60,according to an example embodiment, processes the raw data bynormalizing it to a predefined scale. The above described graphs can bedisplayed alone or together with additional values from other parametersthat are the subject of the experiment, e.g., pH value and flow rate. Inone embodiment, the additional values are displayed on the same graph,e.g., using a different color scheme and superimposed onto thecapacitance values.

Advantageously, the graphical display of the capacitance values allows auser to quickly determine where bubbles are located, and to takeappropriate corrective action in response to the presence of bubbles.The user may decide, for example, to keep those additional values(corresponding to one or more parameters being measured by theexperiment) which are not associated with the locations of detectedbubbles, while discarding values associated with bubble locations.Alternatively, the user may decide that the entire set of data should bediscarded because there are too many bubbles, thus making the additionalvalues unreliable as a whole.

According to an example embodiment, the capacitance values aresuperimposed onto additional measurement data, which additional data isstored in association with layout data representing the physicalconfiguration of the measurement area. The layout data may be stored inan electronic file in the form of an image (e.g., a scanned image of themeasurement area) or text (e.g., a configuration file for a microarrayspotter used to fabricate the array, or a GenePix Array List (GAL)file). The additional measurement data may also be image or text (e.g.,measured pH values stored in a GAL file or measured pH values renderedin grayscale on a scanned image of the measurement area).

According to an example embodiment in which the capacitance values aresuperimposed, a composite display may be generated in step 218, whichdisplay shows a graphical representation of the array together with thecapacitance values superimposed onto the additional measurement valuesat corresponding locations in the array. The superimposition can berendered as text-on-text, text-on-image or image-on-image. An example oftext-on-text is displaying a capacitance value in one half of an arraylocation and additional measurement data in the other half. An exampleof text-on-image is displaying the capacitances using a heat map whilerepresenting the additional measurement data as numerical values on theheat map. An example of image-on-image is displaying the capacitancesusing a heat map while representing the additional measurement datausing a 3-D mesh. Superimposed data may be stored in the electroniclayout file, prior to or in conjunction with the superimposed display.

According to an example embodiment, a processor on the slide or on anexternal computer is configured to automatically invalidate theadditional measurement data (e.g., by replacing measurement values withnull values) in response to detecting bubbles. For example, theprocessor on the slide may detect bubble locations and output anindication of where the bubbles are located to the external computer,which then performs the invalidating based on the indicated locations.This spares the user from having to manually review the capacitancevalues to decide whether to keep the additional measurement data.

According to an example embodiment, bubble detection is combined with pHmodulation. FIG. 26 is a flowchart of a method 300 for pH modulationaccording to an example embodiment of the present invention. Accordingto an example embodiment, the method 300 is performed using the system100.

At step 310, a pair of electrodes that are not currently being used forbubble detection are switched to a pH modulation mode of operation byapplying an input signal, e.g., a pulsed current between the electrodes.Preferably, the switches that control the mode of operation of theelectrodes are implemented using TFTs, e.g., formed using amorphoussilicon, polysilicon or indium gallium zinc oxide (IgZo). An advantageto using thin-film transistors is that the total capacitance of eachelectrode and its corresponding circuitry is reduced, thereby increasingthe speed of measurement in addition to circumventing the need for thickoxides on the electrodes.

To perform bubble detection, an input pulse can be applied, for example,to a single electrode. The input pulse for bubble detection may, butneed not be identical in shape, magnitude or duration to the input pulseused for pH modulation. Changes in capacitance between water contact andbubble contact are detected by observing the electric response of thesame electrode or in the case of mutual capacitance, the response ofanother electrode.

The input signal applied at step 310 for pH modulation may be appliedduring a time in which the input pulse for the bubble detection is notbeing applied. As mentioned above, the input pulse for pH modulation isapplied between a pair of electrodes. This produces a current that,through oxidation and reduction of buffer components (e.g., quinones),changes the pH level of a test solution situated between the electrodes.

At step 312, the input signal is ended before the electrodes are to beused again for bubble detection.

At step 314, the pH level of the test solution is measured to determinewhether additional adjustment is required. Where the slide is configuredfor pH level measurement, the pH level can be calculated at the controlunit 12. Alternatively, the pH level can be measured using a separatetesting device.

At step 316, the input signal is reapplied by the control unit 12 inresponse to determining that further adjustment of the pH level isrequired. In one embodiment, the control unit 12 is configured to applythe input signal multiple times, as a plurality of pH modulating pulses,before determining whether further adjustment is required. The pluralityof pH modulating pulses can be applied to the same pair of electrodes orto a different electrode pair. Similarly, the input signal may bereapplied at step 316 to the same or a different pair of electrodes. Forexample, the pH modulating pulses may be applied to different electrodepairs in a sequential manner so that the entire electrode array istriggered over time to perform pH modulation.

According to an example embodiment, the electrodes can be used toperform functions in addition to bubble detection and pH modulation. Forexample, electrodes can be used for temperature modulation. As anotherexample, the capacitance measurements can be used to estimate thedielectric constant of the test solution, which dielectric constant isthen correlated to a rate of cell growth or a rate with which thesubstance-on-interest binds to a biomolecule.

Example embodiments were described in which the electrodes were arrangedin two layers (FIG. 18 and FIG. 19). However, it will be understood thatthe number of layers can be more or less. In fact, a single layer may besufficient for both pH modulation and bubble detection. Additionally,not every electrode layer needs to be used for pH modulation or bubbledetection. Instead, further electrode layers can be used for otherpurposes, in accordance with the usage of electrodes in conventionalbiosensors.

Example embodiments of the present invention relate to glass slides withat least some of the processing of measurement data being performed onthe slide itself or on a peripheral device connected to a body of theslide, rather than at an external computer responsible for displayingthe processed data. Such slides are referred to herein as aninstrument-on-glass. FIG. 27 to FIG. 29 each show an example embodimentof an instrument-on-glass.

FIG. 27 shows a slide 400 according to an example embodiment of thepresent invention. The slide 400 includes a measurement area 405, apower source 410 and a processing circuit 420. The measurement area 405may be formed of TFTs (for the switches) together with ITO (for theelectrodes). Alternatively, the measurement area 405 may be formed usingonly ITO or ITO in combination with other metals. The power source 410is analogous to the power source 14 in FIG. 14 and may be a battery or apassive power source powered, e.g., using magnetic or RF coupling.

The processing circuit 420 is analogous to the control unit 12 in FIG.14 and may perform preliminary signal processing. Additionally, theprocessing circuit 420 may perform some of the functions describedearlier with respect to the data processing unit 50 (e.g., normalizingor scaling capacitance values or controlling pH modulation). Theprocessing circuit 420 may include a processor (e.g., one or more CMOSchips) that processes the raw data obtained from measurement area 405.The processing circuit 420 may further include a memory storinginstructions or data, used by the processor to process the raw data. Theprocessing circuit 420 may be configured to arrange the raw data into asuitable format for output to an external computer, or to performpreliminary data analysis (e.g., bubble detection and invalidating dataassociated with bubbles). The processor may control the sensingoperation of the measurement area 405 (e.g., driving and reading dataout of the array), perform data compression, and perform wired orwireless transmission of the preliminarily processed data to an externalcomputer. Post-processing and output of the data for display may beperformed at the external computer.

FIG. 28 shows a slide 500 according to an example embodiment of thepresent invention. The components 505, 510 and 520 are analogous to andperform the same functions as the components 405, 410 and 420,respectively, in FIG. 27. However, instead of being located on the bodyof the slide 500, the power source 510 and the processing circuit 520are externally connected, e.g., on a peripheral circuit board 515 thatfits into a hardware interface of the slide 500.

FIG. 29 shows a slide 600 according to an example embodiment of thepresent invention. The components 605, 610 and 620 are analogous to andperform the same functions as the components 405, 410 and 420,respectively, in FIG. 27. In the embodiment of FIG. 29, the power source610 and the processing circuit are externally connected, similar to FIG.28. However, the circuit board 615 includes a serial port connector fortransmission of data and power between the board 615 and the externalcomputer. Specifically, the serial port may be used to transfermeasurement data to the external computer, and to supply power foroperating the measurement area 605 or for recharging the power source610.

An example embodiment of the present invention is directed to one ormore processors, which can be implemented using any conventionalprocessing circuit and device or combination thereof, e.g., a CentralProcessing Unit (CPU) of a Personal Computer (PC) or other workstationprocessor, to execute code provided, e.g., on a non-transitory hardwarecomputer-readable medium including any conventional memory device, toperform any of the methods described herein, alone or in combination,e.g., to output any one or more of the described graphical userinterfaces. The memory device can include any conventional permanentand/or temporary memory circuits or combination thereof, anon-exhaustive list of which includes Random Access Memory (RAM), ReadOnly Memory (ROM), Compact Disks (CD), Digital Versatile Disk (DVD),flash memory, and magnetic tape.

An example embodiment of the present invention is directed to anon-transitory, hardware computer-readable medium, e.g., as describedabove, on which are stored instructions executable by a processor toperform any one or more of the methods described herein.

An example embodiment of the present invention is directed to a method,e.g., of a hardware component or machine, of transmitting instructionsexecutable by a processor to perform any one or more of the methodsdescribed herein.

Example embodiments of the present invention are directed to one or moreof the above-described methods, e.g., computer-implemented methods,alone or in combination.

Example embodiments of the present invention provide devices and methodsfor using electronics to control the pH of a solution close to anelectrode and a device for implementing the methods in an integratedelectronic system. Preferably the devices and methods of the presentinvention are able to control the pH of the solution surrounding theelectrode within a distance of about 1 cm.

According to an example embodiment, a method for changing the pH of asolution by electronic control includes providing an amount of electricinput to the solution using two or more electrodes to electrochemicallygenerate and/or consume hydrogen ions in the solution. The generationand/or consumption of the hydrogen ions are achieved by anelectrochemical reaction of one or more redox active species in thesolution. Preferably, the one or more redox active species is selectedfrom the following: quinones, catechols, aminophenols hydrazines, andderivatives thereof. More preferably, the one or more redox activespecies is a quinone (See Thomas Finley, “Quinones,” Kirk-OthmerEncyclopedia of Chemical Technology, 1-35 (2005)). Even more preferably,the one or more redox active species is selected from the following:hydroquinone, benzoquinone, naphthoquinonenaphthoquinone. Mostpreferably, the one or more redox active species is a quinonederivative, further defined below.

Preferably, the two or more electrodes comprise a sense electrode and areference electrode (RE). In certain example embodiments of the presentinvention, the sense electrode also functions as a working electrode. Incertain example embodiments of the present invention, the two or moreelectrodes include a counter electrode and/or a working electrode. Incertain example embodiments of the present invention, the two or moreelectrodes are each independently made of metal oxide, gold, glassycarbon, graphene, silver, platinum, silver chloride, normal hydrogen,mercury drop, or saturated calomel. In certain example embodiments ofthe present invention, the solution is buffered, unbuffered, aqueous,organic, or a mixture thereof. In certain example embodiments of thepresent invention, the amount of electric input is provided by providingan amount of electric current. In certain example embodiments of thepresent invention, an electric source waveform is selected based on theamount of electric input to be provided. Preferably, the electric sourcewaveform is a galvanostatic waveform or a potentiostatic waveform. Morepreferably, the electric source waveform is selected from apredetermined map that maps electric input amounts to respectivesolution pH values.

According to an example embodiment of the present invention, a methodfor controlling the pH of a solution using two or more electrodesincludes obtaining the open circuit potential (OCP) of two or moreelectrodes in the solution while no electric input is being appliedbetween the two or more electrodes, selecting an amount of electricinput based on the OCP, and providing the amount of electric input tothe solution between the two or more electrodes to change the pH of thesolution. In certain example embodiments of the present invention, theOCP is obtained by measuring the OCP of the two or more electrodes inthe solution or calculated from a known or measured initial pH. Incertain example embodiments of the present invention, the method alsoincludes determining the pH of the solution based on the OCP of the twoor more electrodes in solution. In certain example embodiments of thepresent invention, the method also includes selecting an electric sourcewaveform based on the amount of electric input and the amount ofelectric input is provided according to the selected electric sourcewaveform.

In certain example embodiments of the present invention, the methodfurther includes determining the pH of the solution based on a measuredOCP of the two or more electrodes in the solution. In certain exampleembodiments of the present invention, the amount of electric input isselected based on the determined pH.

According to an example embodiment of the present invention, a methodfor monitoring the pH of a solution using a sense electrode, a referenceelectrode, and a working electrode includes selecting a target opencircuit potential (OCP), characterizing an OCP of the solution betweenthe reference electrode and the sense electrode while no electric inputis being applied to the working electrode, and iteratively performingthe following steps: selecting an amount of electric input to be appliedto the working electrode in order to minimize a difference between theOCP of the solution and the target OCP; and applying the amount ofelectric input to the working electrode to adjust the OCP of thesolution.

In certain example embodiments of the present invention, the target OCPis a fixed value and in other example embodiments, the target OCP is avariable value. In certain example embodiments of the present invention,the target OCP is a range with an upper bound and a lower bound or is asingle value. Preferably, the target OCP is selected based on a targetpH. Preferably, the target pH is user defined. In certain exampleembodiments of the present invention, the sense electrode also functionsas the working electrode. In certain example embodiments of the presentinvention, the sense electrode and the working electrode are distinctelectrodes. Preferably, the distance between the sense electrode and theworking electrode is 0 cm to 1 cm. The electric input may be provided asan electric current or as an electric potential. Preferably, the amountof electric input is provided by applying an electric potential to theworking electrode. More preferably, the electric potential is providedaccording to an electric source waveform. The electric source waveformis a galvanostatic waveform or a potentiostatic waveform. Even morepreferably, the electric source waveform is selected from apredetermined map that maps electric input amounts to respectivesolution pH values. Preferably, the sense electrode is coated with a pHsensitive coating and the OCP of the sense electrode and the pHsensitive coating is dominated by the H⁺ ion concentration. In certainexample embodiments of the present invention, the pH sensitive coatingis an organic material. In other example embodiments of the presentinvention, the pH sensitive coating is an inorganic material.Preferably, the pH sensitive coating is made from a material that isselected from the group consisting of polyaniline, polypyrrole, andiridium oxide.

The example embodiments involve monitoring and/or characterizingelectrochemical parameters and using a detected signal as a feedbackcontrol to generate a desired pH waveform for a specific length of time.All the described methods involve the use of a redox couple to releaseH⁺ ions, lowering pH, upon oxidation, and to consume H⁺ ions, increasingpH, upon reduction based on the formula:RH₂

R+2H⁺+2e ⁻

An example of a redox reaction is the reaction of quinone derivatives.There are many different derivatives of quinone, each with specificoxidative and reductive peaks and the redox reaction rate has been shownto be dependent on the pH of the solution. The electrochemicalproperties of the electrode, including the electron transfer coefficientand the open circuit potential (OCP) in relation to a referenceelectrode, are also important. The OCP has been previously demonstratedto be dependent on the pH of the solution.

According to an example embodiment of the present invention, a devicefor controlling the pH of a solution includes a controller, two or moreelectrodes, and a solution containing one or more redox active species.The device is configured to measure the OCP between the two or moreelectrodes in the solution to generate a measured OCP data and send themeasured OCP data to the controller. The controller is configured toiteratively perform the following steps: select an amount of current oran electric source waveform based on a difference between the target OCPdata and the measured OCP data, apply the selected amount of current tothe solution by providing an electric current or an electric potential,according to the electric potential waveform, to one or more of the twoor more electrodes, and send a request to the device for anothermeasurement of the OCP.

Preferably, the one or more redox active species in the solutiongenerates and/or consumes hydrogen ions through an electrochemicalreaction induced by the electric current or the electric potentialapplied to the solution. Preferably, the one or more redox activespecies is selected from the following: quinone, catechol, aminophenol,hydrazine, and derivatives thereof. More preferably, the one or moreredox active species is a quinone. (See, Thomas Finley, “Quinones,”Kirk-Othmer Encyclopedia of Chemical Technology, 1-35 (2005)). Even morepreferably, the quinone is selected from the following: hydroquinone,benzoquinone, naphthoquinone, and derivatives thereof. Most preferablythe one or more redox active species is a quinone derivative. In certainexample embodiments of the present invention, the solution is buffered,unbuffered aqueous, organic, or a mixture thereof. In certain exampleembodiments of the present invention, the electric source waveform is agalvanostatic waveform or a potentiostatic waveform. Preferably, theelectric source waveform is selected from a predetermined map that mapselectric inputs to respective solution pH values. In certain exampleembodiments of the present invention, the two or more electrodes aremade of metal oxide, glassy carbon, graphene, gold, silver, or platinum.Preferably, the two or more electrodes include a reference electrode anda sense electrode. More preferably, the two or more electrodes furtherinclude a working electrode and/or a counter electrode. Each of the twoof more electrodes is respectively made of metal oxide, gold, glassycarbon, graphene, silver, platinum, silver chloride, normal hydrogen,mercury drop, or saturated calomel. In certain example embodiments ofthe present invention, the sense electrode also functions as a workingelectrode. Preferably, the sense electrode is coated with a pH sensitivecoating and the OCP of the sense electrode and the pH sensitive coatingis dominated by the H+ ion concentration. In certain example embodimentsof the present invention, the pH sensitive coating is an organic or aninorganic material. Preferably, the pH sensitive coating is made from amaterial that is selected the group consisting of polyaniline,polypyrrole, and iridium oxide.

Also provided are quinone derivatives that can be added to solutions andare suitable for electrochemical pH modulation in biological buffers,electrochemically active compositions comprising the quinonederivatives, methods of making the derivatives and/or compositions, anduses thereof.

According to example embodiments, the electrochemically activecomposition comprising a quinone derivative, where the reactivitybetween a nucleophile and the quinone derivative is reduced compared toa reactivity between the nucleophile and an unsubstituted quinone fromwhich the quinone derivative is derived, and the composition isconfigured such that the pH of the composition is electrochemicallymodulated via the quinone derivative. More preferably, the reactivitybetween the nucleophile and the quinone derivative is reduced by atleast 50% compared to the reactivity between the nucleophile and theunsubstituted quinone from which the quinone derivative is derived.Preferably, the quinone derivative is defined by a chemical formulaselected from the group consisting of:

In the above chemical formulas I to XII, each R group is independentlyselected from the group consisting of: H, C_(n)H_(2n+1), Cl, F, I, Br,OM, NO₂, OH, OC_(n)H_(2n), OC_(n)H_(2n)OH, O(C_(n)H_(2n)O)_(y)OH,O(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1), O(C_(n)H_(2n)O)_(y)COOH,O(C_(n)H_(2n)O)_(y)COOM, COOH, COOM, COOC_(n)H_(2n+1),CONHC_(n)H_(2n+1), CON(C_(n)H_(2n+1))₂, SO₃H, SO₃M, NH₂,NHC_(n)H_(2n+1), N(C_(n)H_(2n+1))₂, NHC_(n)H_(2n)OH, NHC_(n)H_(2n)NH₂,N(C_(n)H_(2n)OH)₂, N(C_(n)H_(2n)NH)₂, NHCOC_(n)H_(2n+1),NC_(n)H_(2n+1)COC_(n)H_(2n+1), NC_(n)H_(2n+1)COC_(n)H_(2n)OH,NC_(n)H_(2n+1)COC_(n)H_(2n)NH₂, NC_(n)H_(2n+1)COC_(n)H_(2n)SH, SH,SC_(n)H_(2n), SC_(n)H_(2n)OH, S(C_(n)H_(2n)O)_(y)OH,S(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1), S(C_(n)H_(2n)O)_(y)COOH,S(C_(n)H_(2n)O)_(y)COOM, OC_(n)H_(2n)SH, O(C_(n)H_(2n)O)_(y)SH,O(C_(n)H_(2n)O)_(y)SC_(n)H_(2n+1), C_(n)H_(2n), C_(n)H_(2n)OC_(n)H_(2n),C_(n)H_(2n+1)SC_(n)H_(2n), C_(n)H_(2n)NHC_(n)H_(2n),C_(n)H_(2n)N(C_(n)H_(2n+1))C_(n)H_(2n), C_(n)H_(2n+1), C_(n)H_(2n+1)OH,C_(n)H_(2n+1)OC_(n)H_(2n), C_(n)H_(2n+1)OC_(n)H_(2n)OH,C_(n)H_(2n+1)O(C_(n)H_(2n)O)_(y)COOH,C_(n)H_(2n+1)O(C_(n)H_(2n)O)_(y)COOM, C_(n)H_(2n+1) COOH,C_(n)H_(2n+1)COOM, C_(n)H_(2n+1)COOC_(n)H_(2n+1),C_(n)H_(2n+1)CONHC_(n)H_(2n+1), C_(n)H_(2n+1)CONH(C_(n)H_(2n+1))₂,C_(n)H_(2n+1) SO₃H, C_(n)H_(2n+1) SO₃M, C_(n)H_(2n+1)NH₂,C_(n)H_(2n+1)NHC_(n)H_(2n+1), C_(n)H_(2n+1)N(C_(n)H_(2n+1))₂,C_(n)H_(2n+1)NHC_(n)H_(2n)OH, C_(n)H_(2n+1)NHC_(n)H_(2n)NH₂,C_(n)H_(2n+1)N(C_(n)H_(2n)OH)₂, C_(n)H_(2n+1)N(C_(n)H_(2n)NH₂)₂,C_(n)H_(2n+1)NHCOC_(n)H_(2n+1),C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)OH,C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)NH₂,C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)SH, C_(n)H_(2n+1)SH,C_(n)H_(2n+1)SC_(n)H_(2n), C_(n)H_(2n+1)SC_(n)H_(2n)OH,C_(n)H_(2n+1)S(C_(n)H_(2n+1)O)_(y)OH,C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1),C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)COOH,C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)COOM, sugars, peptides, and amino acids;at least one of the R groups is not hydrogen; M is any metal cation orNH₄ ⁺; n is an integer from 1 to 10⁹; and y is an integer from 1 to 10⁹.

According to example embodiments, all of the R groups of the quinonederivative are different from each other. According to exampleembodiments, two or more of the R groups of the quinone derivative arethe same. Preferably, the composition is an aqueous solution. Accordingto example embodiments, the composition further comprises an additiveselected from the group consisting of: an aqueous buffer, an organicsolvent, an electrolyte, a buffer salt, a bioreagent, a biomolecule, asurfactant, a preservative, a cryoprotectant, and combinations thereof.Preferably the one or more nucleophiles are selected from the groupconsisting of: amines, thiols, amino acids, peptides, proteins, andcombinations thereof. According to example embodiments, the reactivitybetween the nucleophile and the quinone derivative is reduced comparedto the reactivity between the nucleophile and the unsubstituted quinonefrom which the quinone derivative is derived due to: (i) increasedsteric hindrance of a nucleophile binding site by one or more of the Rgroups; (ii) elimination of the nucleophile binding site by covalentbonding between the nucleophile binding site and one of the R groups.Preferably the reactivity between the nucleophile and the quinonederivative is reduced compared to the reactivity between the nucleophileand the unsubstituted quinone from which the quinone derivative isderived due both (i) and (ii).

Also provided are methods comprising modifying a quinone having one ormore R groups by substituting one or more of the R groups with asubstituent to provide a quinone derivative, where the quinonederivative has a reduced reactivity with a nucleophile compared to areactivity between the quinone and the nucleophile; and the substituentis independently selected from the group consisting of: H,C_(n)H_(2n+1), Cl, F, I, Br, OM, NO₂, OH, OC_(n)H_(2n), OC_(n)H_(2n)OH,O(C_(n)H_(2n)O)_(y)OH, O(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1),O(C_(n)H_(2n)O)_(y)COOH, O(C_(n)H_(2n)O)_(y)COOM, COOH, COOM,COOC_(n)H_(2n+1), CONHC_(n)H_(2n+1), CON(C_(n)H_(2n+1))₂, SO₃H, SO₃M,NH₂, NHC_(n)H_(2n+1), N(C_(n)H_(2n+1))₂, NHC_(n)H_(2n)OH,NHC_(n)H_(2n)NH₂, N(C_(n)H₂OH)₂, N(C_(n)H_(2n)NH)₂, NHCOC_(n)H_(2n+1),NC_(n)H_(2n+1)COC_(n)H_(2n+1), NC_(n)H_(2n+1)COC_(n)H_(2n)OH,NC_(n)H_(2n+1)COC_(n)H_(2n)NH₂, NC_(n)H_(2n+1)COC_(n)H_(2n)SH, SH,SC_(n)H_(2n), SC_(n)H_(2n)OH, S(C_(n)H_(2n)O)_(y)OH,S(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1), S(C_(n)H_(2n)O)_(y) COOH,S(C_(n)H_(2n)O)_(y)COOM, OC_(n)H_(2n)SH, O(C_(n)H_(2n)O)_(y)SH,O(C_(n)H_(2n)O)_(y)SC_(n)H_(2n+1), C_(n)H_(2n), C_(n)H_(2n)OC_(n)H_(2n),C_(n)H_(2n)SC_(n)H_(2n), C_(n)H_(2n)NHC_(n)H_(2n),C_(n)H_(2n)N(C_(n)H_(2n+1))C_(n)H_(2n), C_(n)H_(2n+1), C_(n)H_(2n+1)OH,C_(n)H_(2n+1)OC_(n)H_(2n), C_(n)H_(2n+1)OC_(n)H_(2n)OH,C_(n)H_(2n+1)O(C_(n)H_(2n)O)_(y)COOH,C_(n)H_(2n+1)O(C_(n)H_(2n)O)_(y)COOM, C_(n)H_(2n+1)COOH,C_(n)H_(2n+1)COOM, C_(n)H_(2n+1)COOC_(n)H₂₊₁,C_(n)H_(2n+1)CONHC_(n)H_(2n+1), C_(n)H_(2n+1)CONH(C_(n)H_(2n+1))₂,C_(n)H_(2n+1)SO₃H, C_(n)H_(2n+1)SO₃M, C_(n)H_(2n+1)NH₂,C_(n)H_(2n+1)NHC_(n)H_(2n+1), C_(n)H_(2n+1)N(C_(n)H_(2n+1))₂,C_(n)H_(2n+1)NHC_(n)H_(2n)OH, C_(n)H_(2n+1)NHC_(n)H_(2n)NH₂,C_(n)H_(2n+1)N(C_(n)H_(2n)OH)₂, C_(n)H_(2n+1)N(C_(n)H_(2n)NH₂)₂,C_(n)H_(2n+1)NHCOC_(n)H_(2n+1),C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)OH,C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)NH₂,C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)SH, C_(n)H_(2n+1)SH,C_(n)H_(2n+1)SC_(n)H_(2n), C_(n)H_(2n+1)SC_(n)H_(2n)OH,C_(n)H_(2n+1)S(C_(n)H_(2n+1)O)_(y)OH,C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1),C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y) COOH,C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)COOM, sugars, peptides, and amino acids;M is any metal cation or NH₄ ⁺; n is an integer from 1 to 10⁹; and y isan integer from 1 to 10⁹. According to example embodiments, the one ormore R groups are substituted with a polar. Preferably the polar grouphas atoms containing lone pair electrons. Preferably, the polar group iscapable of forming hydrogen bonds with water. Preferably, the polargroup contains at least one of oxygen, nitrogen, and sulfur atoms. Morepreferably, the polar group is selected from the group consisting of:OH, C_(n)H_(2n)OH, OC_(n)H₃, COOH, SO₃H, NH₂, NH₃Cl, ONa, a sugar, anamino acid, and a peptide.

Also provided are methods of synthesizing substituted methyl quinonecomprising: (i) a halide substitution step of reacting a startingmaterial with a hydrogen halide in the presence of acetic acid and analdehyde; (ii) reacting a material produced by step (i) with anucleophile of structure R—X; (iii) reacting a material produced by step(ii) with an oxidizing agent; and (iv) reacting a material produced bystep (iii) with a reducing agent, where R is selected from the groupconsisting of: H, C_(n)H_(2n+1), Cl, F, I, Br, OM, NO₂, OH,OC_(n)H_(2n), OC_(n)H_(2n)OH, O(C_(n)H_(2n)O)_(y)OH,O(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1), O(C_(n)H_(2n)O)_(y)COOH,O(C_(n)H_(2n)O)_(y)COOM, COOH, COOM, COOC_(n)H_(2n+1),CONHC_(n)H_(2n+1), CON(C_(n)H_(2n+1))₂, SO₃H, SO₃M, NH₂,NHC_(n)H_(2n+1), N(C_(n)H_(2n+1))₂, NHC_(n)H_(2n)OH, NHC_(n)H_(2n)NH₂,N(C_(n)H_(2n)OH)₂, N(C_(n)H_(2n)NH)₂, NHCOC_(n)H_(2n+1),NC_(n)H_(2n+1)COC_(n)H_(2n+1), NC_(n)H_(2n+1)COC_(n)H_(2n)OH,NC_(n)H_(2n+1)COC_(n)H_(2n)NH₂, NC_(n)H_(2n+1)COC_(n)H_(2n)SH, SH,SC_(n)H_(2n), SC_(n)H_(2n)OH, S(C_(n)H_(2n)O)_(y)OH,S(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1), S(C_(n)H_(2n)O)_(y)COOH,S(C_(n)H_(2n)O)_(y)COOM, OC_(n)H_(2n)SH, O(C_(n)H_(2n)O)_(y)SH,O(C_(n)H_(2n)O)_(y)SC_(n)H_(2n+1), C_(n)H_(2n), C_(n)H_(2n)OC_(n)H_(2n),C_(n)H_(2n)SC_(n)H_(2n), C_(n)H_(2n)NHC_(n)H_(2n),C_(n)H_(2n)N(C_(n)H_(2n+1))C_(n)H_(2n), C_(n)H_(2n+1), C_(n)H_(2n+1)OH,C_(n)H_(2n+1)OC_(n)H_(2n), C_(n)H_(2n+1)OC_(n)H_(2n)OH,C_(n)H_(2n+1)O(C_(n)H_(2n)O)_(y)COOH,C_(n)H_(2n+1)O(C_(n)H_(2n)O)_(y)COOM, C_(n)H_(2n+1)COOH,C_(n)H_(2n+1)COOM, C_(n)H_(2n+1)COOC_(n)H_(2n+1),C_(n)H_(2n+1)CONHC_(n)H_(2n+1), C_(n)H_(2n+1)CONH(C_(n)H_(2n+1))₂,C_(n)H_(2n+1)SO₃H, C_(n)H_(2n+1)SO₃M, C_(n)H_(2n+1)NH₂,C_(n)H_(2n+1)NHC_(n)H_(2n+1), C_(n)H_(2n+1)N(C_(n)H_(2n+1))₂,C_(n)H_(2n+1)NHC_(n)H_(2n)OH, C_(n)H_(2n+1)NHC_(n)H_(2n)NH₂,C_(n)H_(2n+1)N(C_(n)H_(2n)OH)₂, C_(n)H_(2n+1)N(C_(n)H_(2n)NH₂)₂,C_(n)H_(2n+1)NHCOC_(n)H_(2n+1),C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)OH,C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)NH₂,C_(n)H_(2n+1)NC_(n)H_(2n+1)COC_(n)H_(2n)SH, C_(n)H_(2n+1)SH,C_(n)H_(2n+1)SC_(n)H_(2n), C_(n)H_(2n+1)SC_(n)H_(2n)OH,C_(n)H_(2n+1)S(C_(n)H_(2n+1)O)_(y)OH,C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)OC_(n)H_(2n+1),C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)COOH,C_(n)H_(2n+1)S(C_(n)H_(2n)O)_(y)COOM, sugars, peptides, and amino acids;M is any metal cation or NH₄ ⁺; n is an integer from 1 to 10⁹; y is aninteger from 1 to 10⁹; and X is either OH, NH₂, NHR, SH, O⁻, or S⁻.

According to example embodiments, the starting material isdialkoxybenzene and the result of the halide substitution step is anortho-quinon, para-quinone, or a combination thereof. Preferably, thenumber of halide groups per molecule of the ortho-quinone orpara-quinone is 1, 2, 3, or 4. According to example embodiments, thestarting material is dialkoxynaphthalene and the result of the halidesubstitution step is an ortho-naphthoquinone, para-naphthoquinone, or acombination thereof. Preferably, the number of halide groups permolecule of the ortho-naphthoquinone or para-naphthoquinone is 1 or 2.Preferably, the hydrogen halide is selected from the group consistingof: HCl, HBr, HI, and combinations thereof. Preferably, the oxidizingagent is selected from the group consisting of: cerium ammonium nitrate,iodine, hydrogen peroxide, hypervalent iodine, iodobenzene diacetate,bromine compounds, and combinations thereof. Preferably, the reducingagent is selected from the group consisting of: sodium borohydrate,potassium borohydrate, sodium hydrosulfite, trichlorosilane, andcombinations thereof.

Also provided are methods comprising: providing a biosensor comprising asupport in an solution, the support comprising one or more electrodesand a biomolecule interface layer having one or more immobilized probesthereon, and the solution comprising a quinone derivative; adding abiomolecule analyte to the solution; electrochemically reacting thequinone derivative using the one or more electrodes to produce an amountof H⁺ ions and/or an amount of OH⁻ ions, wherein the pH of the solutionclose to the one or more electrodes is controlled by the amount of H⁺ions and/or the amount of OH⁻ ions produced; collecting signals from thebiosensor, where a reactivity between a nucleophile and the quinonederivative is reduced compared to a reactivity between the nucleophileand an unsubstituted quinone from which the quinone derivative isderived

Preferably, the pH of the solution before electrochemically reacting thequinone derivative using the one or more electrodes is 1 to 14.Preferably, the pH of the solution after electrochemically reacting thequinone derivative using the one or more electrodes is 1 to 14.Preferably, the solution contains one or more nucleophiles. Preferably,the one or more nucleophiles are selected from the group consisting of:amines, thiols, amino acids, peptides, proteins, and combinationsthereof. According to example embodiments, the solution contains areduced quinone derivative and electrochemically reacting the quinonederivative results in an electrochemical oxidation reaction of thereduced quinone derivative to make the pH of the solution more acidic.Preferably, the concentration of the reduced quinone derivative is 0 to1M. According to example embodiments, the solution contains an oxidizedquinone derivative and electrochemically reacting the quinone derivativeresults in an electrochemical reduction reaction of the oxidized quinonederivative to make the pH of the solution more basic. Preferably, theconcentration of the oxidized quinone derivative is 0 to 1M. Preferably,the solution contains one or more buffer components provided in aconcentration that is 0 to 1M. Preferably, the one or more buffercomponents are selected from the group consisting of: organic solvents,electrolytes, bioreagents, biomolecules, surfactants, and combinationsthereof. According to example embodiments, the method further comprisesmeasuring the pH of the solution. Preferably, the pH is measuredcontinuously. Preferably, the quinone derivative is electrochemicallyreacted by providing an amount of electric current. Preferably, the pHis measured before providing the amount of electric current. Preferably,the amount of electric current is selected based on the measured pH.

Quinones are herein defined as unsubstituted quinones. For example, thechemical structures I-XII would represent quinones if all of the Rgroups were hydrogen. Quinone derivatives are herein defined ascompounds that are structurally similar to quinones except that at leastone hydrogen is replaced by a substituent. In cases where more than onehydrogen is replaced by a substituent, the identity of the substituentsmay be independently selected but are not required to be unique.Compared to their unsubstituted counterparts the quinone derivatives ofthe present invention have reduced reactivity with nucleophiles and thepH of the biological buffer containing the quinone derivatives is ableto be electrochemically modulated.

Also provided are methods for controlling a pH of a solution using twoor more electrodes, the method comprising measuring an open circuitpotential (OCP) of the two or more electrodes in the solution while nocurrent is being applied between the two or more electrodes, selectingan amount of current based on the measured OCP, and providing theselected amount of current to the solution, thereby changing the pH ofthe solution by at least one electrochemically generating or consuminghydrogen ions. Preferably, generation and/or consumption of the hydrogenions are achieved by an electrochemical reaction of one or more redoxactive species in the solution. Preferably, the one or more redox activespecies is selected from the group consisting of: quinones, catechols,aminophenols, hydrazines, derivatives thereof, and combinations thereof.Preferably, the one or more redox active species is a quinone selectedfrom the following: hydroquinone, benzoquinone, naphthaquinone,derivatives thereof, and combinations thereof. Preferably, the two ormore electrodes comprise a sense electrode and a reference electrode.According to example embodiments, the sense electrode is configured toalso function as a working electrode. Preferably, the two or moreelectrodes also comprise a counter electrode and/or a working electrode.Preferably, the two or more electrodes are each independently made of amaterial selected from the group consisting of: metal oxide, gold,glassy carbon, graphene, silver, platinum, silver chloride, normalhydrogen, mercury drop, saturated calomel, and combinations thereof.Preferably, the solution is buffered, unbuffered, aqueous, organic, or amixture thereof. Preferably, the method further comprises determiningthe pH of the solution based on the measured OCP of the two or moreelectrodes in the solution. Preferably, selection of an electricalwaveform is based on the determined pH. Preferably, the method furthercomprises selecting an electrical waveform and providing the electricalwaveform to the solution. The electrical waveform is either agalvanostatic waveform or a potentiostatic waveform. Preferably, theelectrical waveform is selected from a predetermined map that mapsrespective current amounts to respective electrical waveforms.

Also provided is a biosensor system comprising a support that includesan a sense electrode, a reference electrode, and a working electrode; anelectrochemically active agent, wherein the biosensor is configured tocontrol a change of a redox state of the electrochemically active agent,and the biosensor is configured to iteratively perform the following:selecting an amount of current to be applied to the working electrode inorder to minimize a difference between the OCP of the solution and thetarget OCP; applying the selected amount of current to the workingelectrode to adjust the OCP of the solution; and measuring the OCP ofthe solution. Preferably, the solution is an aqueous solution. Accordingto example embodiments, the sense electrode also functions as theworking electrode. According to example embodiments, the sense electrodeand the working electrode are distinct electrodes and a distance betweenthe sense electrode and the working electrode is 0 cm to 1 cm.Preferably, the amount of current applied to the working electrode isprovided by applying an electrical waveform. The electrical waveform isa galvanostatic waveform or a potentiostatic waveform. Preferably, theelectrical waveform is selected from a predetermined map that mapsrespective current amounts to respective electrical waveforms.Preferably, the sense electrode is coated with a pH sensitive coating.The pH sensitive coating is an organic material or an inorganicmaterial. Preferably, the pH sensitive coating is made from a materialselected from the group consisting of: polyaniline, polypyrrole, iridiumoxide, and a combination thereof.

Also provided are methods for monitoring pH of a solution using a senseelectrode, a reference electrode, and a working electrode, the methodcomprising selecting a target open circuit potential (OCP) based on atarget pH for the solution; characterizing an OCP of the solutionbetween the reference electrode and the sense electrode while no currentis being applied to the working electrode; and iteratively performingthe following: selecting an amount of current to be applied to theworking electrode in order to minimize a difference between the OCP ofthe solution and the target OCP; applying the selected amount of currentto the working electrode to adjust the OCP of the solution; andmeasuring the OCP of the solution. Preferably, the solution is anaqueous solution. Preferably, the target pH is set by incorporating anelectro-chemical delta-sigma-modulator. More preferably, theoutput-signal of the electro-chemical delta-sigma-modulator is digitallyfiltered to get a digital representation of the charge needed to createthe target pH. Preferably, the target OCP is a range with an upper boundand a lower bound. More preferably, the target pH is a single targetvalue. According to example embodiments, the sense electrode alsofunctions as the working electrode. According to example embodiments,the sense electrode and the working electrode are distinct electrodesand a distance between the sense electrode and the working electrode is0 cm to 1 cm. Preferably, the amount of current applied to the workingelectrode is provided by applying an electrical waveform. The electricalwaveform is a galvanostatic waveform or a potentiostatic waveform.Preferably, the electrical waveform is selected from a predetermined mapthat maps respective current amounts to respective electrical waveforms.Preferably, the sense electrode is coated with a pH sensitive coating.The pH sensitive coating is an organic material or an inorganicmaterial. Preferably, the pH sensitive coating is made from a materialselected from the group consisting of: polyaniline, polypyrrole, iridiumoxide, and combinations thereof.

Also provided is a device for controlling a pH of a solution comprising:a controller; two or more electrodes; and a solution containing one ormore redox active species, wherein the device is configured toiteratively perform the following: measure an open circuit potential(OCP) between the two or more electrodes in the solution to generate ameasured OCP data; select, using the controller, an amount of current oran electric potential waveform based on a difference between target OCPdata and the measured OCP data; and apply, using the controller, theselected amount of current or the selected electric potential waveformto the solution via one or more of the two or more electrodes.Preferably, the solution is an aqueous solution. Preferably, the one ormore redox active species generates or consumes hydrogen ions through anelectrochemical reaction induced by the electric current or the electricpotential applied to the solution. Preferably, the one or more redoxactive species is selected from the following: quinones, catechols,aminophenols hydrazines, derivatives thereof, and combinations thereof.Preferably, the one or more redox active species is a quinone selectedfrom the following: hydroquinone, benzoquinone, naphthaquinone,derivatives thereof, and combinations thereof. Preferably, the solutionis buffered, unbuffered aqueous, organic, or a mixture thereof.Preferably, the electric potential waveform is a galvanostatic waveformor a potentiostatic waveform. More preferably, the electric potentialwaveform is selected from a predetermined map that maps respectivecurrent amounts to respective electric potential waveforms. Preferably,the two or more electrodes comprise two or more of a referenceelectrode, working electrode, counter electrode, or sense electrode thatis made of a material independently selected from the group consistingof: metal oxide, gold, glassy carbon, graphene, silver, platinum, silverchloride, normal hydrogen, mercury drop, saturated calomel, andcombinations thereof. According to example embodiments, the senseelectrode also functions as a working electrode. Preferably, the senseelectrode is coated with a pH sensitive coating. The pH sensitivecoating is an organic material or an inorganic material. Preferably, thepH sensitive coating is made from a material selected from the groupconsisting of: polyaniline, polypyrrole, iridium oxide, and combinationsthereof.

A drawing of a system having electrochemically active agent in solution,according to an example embodiment of the present invention, ispresented in FIG. 30. In previously described systems, theelectrochemically active agent was attached to the electrode surface andnot in solution. As shown in FIG. 30, the system provides an anodeelectrode and an electrochemically active agent in solution. Applying acurrent to the electrode induces the electrochemically active agent toundergo an electrochemical redox reaction which makes the pH of thesolution near the electrode more acidic. Having the electrochemicallyactive agent in solution rather than attached to the electrode surfacehas many advantages. For example, a more significant change can beinflicted on the surrounding environment if the amount ofelectrochemically active agent is not limited by the density of thesurface layer, thereby increasing capacity of the device; freshelectrochemically active agent can be supplied to the electrode surfacevia diffusion from bulk solution, thereby allowing for cyclingcapability; and universal electrochemistry can be applied to all typesof electrodes, which will not interfere with other surface chemistriessuch as attachment of anti-fouling reagents or biomolecules.Furthermore, in order to make use of quinones as electrochemicallyactive agents for pH generation in biological solutions, the structureof the quinones were modified to satisfy the requirements for use inbiological solutions. In order to be useful for pH modulation inbiological buffers, a molecule should satisfy the followingrequirements: release or consume protons through electrochemicalreaction upon electronic stimulation, sufficient water solubility,reduction and oxidation potential should be lower than the potential ofwater hydrolysis or other redox active species within the solution,stability in solution in the absence of electronic stimulation (i.e., noautooxidation/reduction), low reactivity towards nucleophiles,compatibility with biological samples (for example: proteins, peptides,cells, DNA, and enzymes).

FIG. 31 shows quinone derivatives that can be used for pH modulation inaqueous solutions, according to example embodiments of the presentinvention.

The above description is intended to be illustrative, and notrestrictive. Those skilled in the art can appreciate from the foregoingdescription that the present invention may be implemented in a varietyof forms, and that the various embodiments can be implemented alone orin combination. Therefore, while the embodiments of the presentinvention have been described in connection with particular examplesthereof, the true scope of the embodiments and/or methods of the presentinvention should not be so limited since other modifications will becomeapparent to the skilled practitioner upon a study of the drawings,specification, and appendices. Further, steps illustrated in theflowcharts may be omitted and/or certain step sequences may be altered,and, in certain instances multiple illustrated steps may besimultaneously performed.

The following are examples which illustrate specific methods without theintention to be limiting in any manner. The examples may be modifiedwithin the scope of the description as would be understood from theprevailing knowledge.

Related applications International Patent App. No. PCT/EP2015/052661 andU.S. Pat. Provisional App. Ser. No. 61/939,396 are hereby incorporatedby references in their entirety.”

EXAMPLES Example 1—Electrochemical Generation of H+ or OH− Ions atElectrode Surfaces

Electrode material used: The electrode material was indium tin oxide.This is a semiconducting electrode surface with very large potentialwindow in an aqueous solution.

Electro-Oxidation of Species to Produce H⁺ Ions.

Oxidation of ascorbic acid at the electrode surfaces produced H⁺ ionsand changed the electrode surface pH to a more acidic state:AH₂→A+2H⁺+2e ⁻,where AH₂ is ascorbic acid (C₆H₆O₆) (as shown in FIG. 5). The electrodepotential at which it oxidizes was less than 0.5V for Indium tin oxidematerial vs Ag/AgCl reference electrode (as shown in FIG. 7). Thispotential was less than the voltages needed for the oxygen evolutionreaction in aqueous solution. Higher electrode potential (e.g >1V forITO electrodes in just phosphate buffer) can damage the PEG layer (asshown in FIG. 8). The ascorbic acid also acted as a sacrificial speciesto prevent electrochemical degradation of the surface chemistry.

Electro-Reduction of Species to Produce OH− Ions.

Reduction of benzoquinone (C₆H₄O₂) into Hydroquinone (C₆H₆O₂) canproduce OH⁻ ions at −0.1V:BQ+2e ⁻+2H₂O→HQ+2OH⁻This reduction reaction increased the pH at the electrode interface.

In the above examples the amount of H⁺ or OH⁻ ions generated will dependon the concentration of species present in solution (nM-mM range),potential applied (−2V to +2V), type of waveform (pulse, constant,sawtooth, sinusoidal, square wave at different frequencies and dutycycles), and diffusion of the species (can be varied due to additives inthe solution). These parameters can be optimized to get different pHs atthe each of the electrode element present in the multisite biosensor.

Example 2—pH Change Using Enzymatic Reactions

Enzymes such as oxidases, ureases or dehydrogenases have been known toconsume or generate hydrogen during the reaction. For example:β-d-glucose+O₂ →d-glucose-δ-lactone+H₂O₂d-glucose-δ-lactone+H₂O→d-gluconate+H⁺Oxidation of glucose in the presence of glucose oxidase can produce H⁺ions that are used to change the pH near the proteins of interest.

Example 3—Co-Immobilization of Enzymes Along with Biomolecular Probes ina Biomolecular Interface Layer

The enzymes when co-immobilized on the surface along with proteinsbrings them in close proximity so the H⁺ produced by the enzymaticreaction will lead to a localized pH change that can affect proteinbinding (for example antigen-antibody binding and non-specific binding).

Example 4—Attaching the Enzymes to Magnetic Micro/Nanoparticles

Proteins are attached to micro/nanocavities of a solid surface on anelectromagnet. The enzymes are separately attached to magneticmicro/nanoparticles in the solution. Controlling the electromagnet thatis fabricated/placed underneath controls the local pH values. Then theenzymatic reaction is triggered by introducing the corresponding enzymesubstrate (as shown in FIG. 6). Alternatively electrochemically activeenzymes are used. The pH change is localized on the cavities and theprotein interactions are modulated.

Example 5—Electrochemical Modulation of pH as Monitored by FluorescenceIntensity with Green Fluorescence Protein (GFP)

Electrode material used: The electrode material was indium tin oxide.The fluorescent protein used is GFP immobilized on a glass substratewhich includes an array of electrodes. The GFP is applied as spots, eachspot covers an area that overlaps with one electrode and an area that isnot overlapping with an electrode.

The pH change at the surface of ITO working electrode is generated viacurrent-driven oxidation of a redox active molecule,2-methyl-1,4-dihydroquinone, in diluted phosphate buffer (pH=7.4)containing 0.1M Na₂SO₄. After 10 seconds of induction, current (50microamps) was applied for 30 second, which resulted in a drop ofsolution pH to 5.5, as was observed by a change in GFP fluorescenceintensity. FIG. 10 is used as calibration curve to assess the pH values.After current was turned off, the pH recovered to neutral value within50 seconds (as shown in FIGS. 11 and 12B).

Example 6—Preventing Reaction with Nucleophiles

Para-benzoquinones and ortho-benzoquinones (compounds 2, 4, 6, and 9 inFIG. 31) are susceptible to nucleophilic attack at the double bond ofthe ring (positions 2, 3, 5 and 6 in structure 2 of FIG. 31, positions3, 4, 5 and 6 in structure 4 of FIG. 31, and positions 2 and 3 instructure 6 of FIG. 31). Introducing substituents at some or all ofthose positions can mitigate the problem of nucleophilic attack. Forexample, 1,4-benzoquinone (structure 2 in FIG. 31, where R1, R2, R3 andR4 are H) undergoes Michael addition reaction with amino groups ofproteins (Loomis et al., Phytochemistry, 5, 423, (1966) and U.S. Pat.No. 6,753,312 B2). On the other hand, 2,5-disubstituted1,4-benzoquinones (structure 2 in FIG. 30, where R1 and R3 are H, and R2and R4 are groups other than H) do not show susceptibility to Michaeladdition reaction in the presence of proteins and are more suitable foruse in biological buffers. FIG. 32 demonstrates the effect ofsubstitution on protein stability in the presence of benzoquinones.Fluorescence intensity of Green Fluorescent Protein (GFP) was measuredafter incubation with three different benzoquinones in phosphatebuffered saline for 30 min (concentration of benzoquinones was 0.5 mM).The difference in fluorescence intensity indicates the varied effectthat the different substitutions in benzoquinones have on the stabilityof GFP. Fluorescence intensity of GFP is indicative of its structuralintegrity. Losses in fluorescence intensity usually indicate loss of itstertiary structure (protein denaturation) (Yin D. X., Zhu L., Schimke R.T. Anal. Biochem. 1996, 235:195-201). As shown in FIG. 32, GFP retains100% of its fluorescence intensity after incubation with di-substitutedbenzoquinone for 30 min, while incubation with unsubstitutedbenzoquinone and mono-substituted benzoquinone causes 25% and 7% loss influorescence intensity, respectively.

Example 7—Tuning Water Solubility

Water solubility of aromatic compounds can be improved by introducingcharged groups or atoms with lone pair electrons that can participate inhydrogen bonding. Such groups are, for example, —OH, —C_(n)H_(2n)OH,—OC_(n)H₃, —COOH, —SO₃H, —NH₂, —NH₃Cl, —ONa. Sugars, amino acids, andpeptides can also improve water solubility of quinones. Syntheticmacromolecules such as polyethyleneglycoles can be used as substituentsas well.

Example 8—Adjusting Redox Window

Reduction/oxidation potential of quinones can be tuned to fit the needsof specific application. By introducing electron-donating groups (suchas alkyl, hydroxyl, alkoxy, methoxymethyl, morpholinomethyl, amino andchloro substituents) redox potential can be pushed towards highervoltage. Conversely, electron-withdrawing groups (such as nitro, cyano,carboxylic acid or carboxylic ester groups) will push redox potentialtowards lower voltage.

In an example embodiment, the mechanism of oxidation of hydroquinones inaqueous solutions involves two steps: transfer of electrons and transferof protons. Introducing electron-withdrawing or electron-donatingsubstituents addresses the first step of the process. Oxidationpotential of hydroquinones can also be lowered by introducingsubstituents that are capable of forming intramolecular hydrogen bondswith hydroxyl groups of hydroquinone. Such hydrogen bonding weakens thebond between hydrogen and oxygen of hydroxyl group, therefore loweringan overall energy barrier for oxidation reaction. Examples of such Rgroups are C_(n)H_(2n)OH, C_(n)H_(2n)OC_(n)H₃, morpholinomethyl, andCOOC_(n)H₃.

Having the ability to select molecules which undergo electrochemicaltransformation through the same mechanism, but at different potentialsenables one to accommodate different pH conditions and avoid undesirableelectrochemical reactions involving other redox active components in thesystem. For example, for applications involving DNA synthesis, it isimportant to keep the voltage below the reduction potentials ofpyrimidine bases, nucleosides, and nucleotides (1V vs. NHE in aqueoussolution pH 8) (Steenken, S. J Am Chem Soc, 1992, 114: 4701-09).

Autooxidation of hydroquinones is another issue. In an exampleembodiment, in order to avoid oxidation, a quinone derivative with highenough electrochemical oxidation potential that is resistant tospontaneous chemical oxidation by molecular oxygen is used.

Conversely, benzoquinones with low enough reduction potentials should bechosen for systems where reducing agents, like mercaptoethanol,glutathione, and dithiothreitol are present in solution. Examples ofsuch applications are DNA synthesis, electrophoresis, and immunoassays.

The redox potential of quinones is affected by the pH of the solution.It is easier to oxidize hydroquinones in more basic pH, whereas moreacidic pH will require higher oxidation potentials. This, in turn,affects stability of hydroquinones towards autooxidation by oxygen inair. Therefore, if one needs to work in basic pH, using a quinone withhigher oxidation potential will improve the stability of electrochemicalsystem. FIG. 33 shows that there are different oxidation potentials fordifferent substituents in quinones (FIG. 33 A-D), and therefore, theoxidations potential can be tuned by varying the substituents inquinones.

Example 9—Synthesis of Substituted Quinones

Scheme 1 shown in FIG. 34 is a representation of a synthesis ofsubstituted quinone according to an example embodiment of the presentinvention.

Example 10—2,5-dimethyl-1,4-hydroquinone

Sodium dithionate (18.7 g, 107.3 mmol, 7.3 equiv) was dissolved in 20 mLH₂O and loaded into a separatory funnel. Next, a solution ofbenzoquinone (2 g, 14.7 mmol, 1 equiv) in 75 mL diethyl ether was added.The diphasic mix was stirred vigorously for 30 minutes and the organiclayer changed color from orange to pale yellow. Organic phase was washedwith brine, dried over MgSO₄, and concentrated to yield a white solid(1.69 g, 83%).

Example 11—1,4-Bis(bromomethyl)-2,5-dimethoxybenzene

Paraformaldehyde (Aldrich, 4.27 g, 144.75 mmol) and HBr/AcOH (Fluka,33%, 30 mL) were added slowly to a stirred solution of1,4-dimethoxybenzene (Aldrich, 10.00 g, 72.37 mmol) in glacial aceticacid (Fisher, 50 mL). The mixture was stirred at 50° C. for one hour,allowed to cool to room temperature, and then hydrolyzed in water (200mL). The white solid was collected by filtration, suspended in C_(n)HCl₃(50 mL), and refluxed for 10 min. After cooling to room temperature, thewhite solid was again collected by filtration and washed with water(15.75 g, 67%). NMR spectra were obtained experimentally to confirm thechemical structure of the resulting compound and its purity. NMR resultswere as follows: ¹H NMR (300 MHz, CDCl₃) δ: 6.88 (s, 2H), 4.54 (s, 4H),3.87 (s, 6H) ppm.

Example 12—1,4-dimethoxy-2,5-dimethoxymethylbenzene

A dry round bottom flask was charged with2,5-dibromomethyl-1,4-dimethoxybenzene (3 g, 9.26 mmol, 1.0 equiv),anhydrous K₂CO₃ (25.6 g, 185 mmol, 20 equiv), and dry methanol (200 mL).The reaction mixture was heated to reflux for 30 min, then cooled toambient temperature, filtered, and concentrated to a crude white solid.The solid was resuspended in water and extracted with ethyl acetate,dried over MgSO₄, and concentrated. The residue was recrystallized inhexanes as a pale yellow powder (1.2 g, 57%). Retention factor (Rf) (50%EtOAc/hexanes)=0.65.

Example 13—2,5-dimethoxymethyl-1,4-benzoquinone

A solution of 1,4-dimethoxy-2,5-dimethoxymethylbenzene (1.2 g, 5.24mmol, 1.0 equiv) in acetonitrile (0.1 M, 52 mL) was treated with asolution of cerium ammonium nitrate (5.8 g, 10.6 mmol, 2.02 equiv) inwater (8 mL). The reaction mixture was stirred under argon at ambienttemperature for 30 minutes, then diluted with water, and extracted withdichloromethane. The combined organic layers were washed with water,dried over Na₂SO₄, and concentrated to an orange solid. The crude mixwas purified by column chromatography on deactivated silica gel (0-5%ethyl acetate/hexanes) to yield yellow crystals (300 mg, 67%). Rf (50%EtOAc/hexanes)=0.7; ¹H NMR (300 MHz, CDCl₃) δ: ppm; UV-Vis=268 nm.

Example 14—2,5-dimethoxymethyl-1,4-hydroquinone

The benzoquinone obtained according to the reaction of Example 13 (200mg, 1.02 mmol, 1.0 equiv) in 2.5 mL EtOAc was treated with a solution ofsodium dithionate (1.3 g, 7.44 mmol, 7.3 equiv) in 2 mL H2O. Thediphasic mix was stirred vigorously for 30 minutes and the solutionchanged colors from bright to pale yellow. The mixture was diluted withwater, extracted with ethyl acetate, dried over MgSO₄, and concentratedto a white powder (96 mg, 48%).

Example 15—1,4-dimethoxy-2,5-dihydroxymethylbenzene

A dry round bottom flask was charged with2,5-dibromomethyl-1,4-dimethoxybenzene (5 g, 15.4 mmol, 1.0 equiv) andNaOH (77 mL of 1.0 M solution, 77 mmol, 5.0 equiv), 12 mL H2O, and 38 mLTHF. The reaction mixture was sealed and heated to 80° C. for 6 h. Aftercooling, the reaction mixture was concentrated by rotary evaporation toa crude solid that was recrystallized in hexanes to a white powder (3 g,60%). Rf (80% EtOAc/hexanes)=0.2.

Example 16—2,5-dimethoxymethyl-1,4-benzoquinone

A solution of 1,4-dimethoxy-2,5-dihydroxymethylbenzene (1.0 g, 5.04mmol, 1.0 equiv) in acetonitrile (0.2 M, 25 mL) was treated with asolution of cerium ammonium nitrate (5.5 g, 10.1 mmol, 2.0 equiv) inwater (33 mL) at 0° C. The reaction mixture was stirred under argon atambient temperature for 30 min, and then extracted with EtOAc. Thecombined organic layers were dried over Na₂SO₄ and concentrated to anorange-red solid. The crude mix was purified by column chromatography ondeactivated neutral alumina (0-100% ethyl acetate/hexanes) to yieldyellow crystals (300 mg, 67%). Rf (80% EtOAc/hexanes)=0.4; ¹H NMR (DMSO,500 MHz): 6.6 (s, 2H), 5.3 (s, 2H), 4.3 (s, 4H), ppm; ¹³C NMR (DMSO, 125MHz): 187.4, 149.1, 129.8, 57.0 ppm; UV-Vis=260 nm.

Example 17—2,5-dihydroxymethyl-1,4-hydroquinone

The benzoquinone obtained according to the reaction of Example 16 (60mg, 0.36 mmol, 1.0 equiv) in 0.6 mL EtOAc was treated with a solution ofsodium dithionate (453 mg, 2.6 mmol, 7.3 equiv) in 0.7 mL H₂O. Thediphasic mix was stirred vigorously for 30 minutes and the solutionchanged colors from bright to pale yellow. The mixture was diluted withwater, extracted with ethyl acetate, dried over MgSO₄, and concentratedto a crude solid that was purified by column chromatography ondeactivated neutral alumina under Ar to yield a white powder (26 mg,30%). UV-Vis=297 nm.

Example 18—Open Loop Method

According to an example embodiment, a method, termed open loop pHcontrol, involves using electric current or electric potential shapingto maintain a desired pH of a solution close to the electrode. Themethod relies on an understanding of the electrochemical components ofthe system, the major constituents being the reduction/oxidationproperties of the quinone, the starting pH of the solution, the electrontransfer coefficient of the electrode material, the redox moleculeconcentration, the salt concentration, and the buffer composition andconcentration. All these components impact how the electrochemicalreaction changes the pH close to the electrode. With this understandingand by incorporating experimental data, a series of models can be usedto define the waveforms to change the pH as required.

FIG. 35 shows an outcome from an open loop waveform experiment designedto hold the pH of the solution close to the electrode at pH 6.5 over 15minutes. The stepped trace approximately extending between currentvalues 3.12e⁻⁶ and 3.50e⁻⁶ correlates with the current applied to thesystem (right axis). The approximately straight trace correlates withthe observed pH close to the electrode surface, which is measured byanalyzing the pH dependent fluorescence of green fluorescent proteinbound to the surface (left axis).

FIG. 36 shows four different examples of waveforms (A-E) usable to shapeand control the pH of the solution. The black line is thecurrent/potential driven input and the grey line is the resultant pHchange in the solution close to the electrode surface plotted over time.

If the relevant electrochemical components of a system remain fixed,these waveforms can be used to reproducibly generate the same pH changeprofile of the solution on demand without additional complexity. Themethod can be implemented using various electrode systems, schematics ofexample setups are shown in FIG. 40A-B. The method requires a minimum of2 electrodes 815, 820. Initially, the open circuit potential of thesolution close to the electrode 700 can be measured by applying zerocurrent between the 2 electrodes 815, 820. In this state, one electrodeacts as the sense electrode (SE) 815 and the second acts as a referenceelectrode (RE) 820. Once the starting OCP is known a current 800 (FIG.40A) or an electric potential 804 (FIG. 40B) is applied to theelectrodes 815, 820 based on the desired pH change. While a current orpotential is being applied, one electrode 815 acts as a workingelectrode (WE) and the second electrode 820 acts as a counter electrode(CE). This is the simplest case in that the conditions of the systemmust remain fixed, but to improve accuracy, a method for continuousfeedback of the system state is preferred.

Example 19—Closed Loop Method

According to another example embodiment, a method, termed closed loop pHcontrol, uses the open circuit potential as a feedback measurement tocontrol the current or potential.

FIG. 38A, illustrates a controlled OCP on the SE by using a closed loopfeedback method with a single OCP V_(TARGET). In one setup (shown inFIG. 41), the system is driven to apply a current from a current source800 to increase the H⁺ concentration in solution 700 until the V_(in)830 detected by the SE 817 reaches a single OCP V_(TARGET) value. Thencurrent source 800 is shut off using switch 802 and diffusion of the H⁺ions away from the SE 817 results in reduction of the H⁺ concentrationin solution 700. Similarly, the system can be set to drive a decrease inthe H⁺ concentration until a OCP V_(TARGET) value is reached and thendiffusion of the H⁺ ions towards the SE results in increase of the H⁺concentration (not shown). In another setup (shown in FIG. 42), positive800 and negative 802 current sources are used so that the system doesnot need to rely on diffusion to facilitate the change of pH. Thissystem can actively change the pH in both a positive and negativedirection. In this setup the system is driven to apply a positivecurrent from a current source 800 to increase the H⁺ concentration untilthe V_(in) 830 reaches a single OCP V_(TARGET) value and a changeoverswitch 803 is used to connect a negative current source 801 to the WE816 to apply a negative current and drive the setup in reverse to reducethe H⁺ concentration. This way, the system does not rely on passivediffusion but actively monitors and adjusts the pH by electroniccontrol.

FIG. 38 B shows experimental data of the OCP between the SE 817 and RE822, which changes as current is applied to the WE 816. In this setup(shown in FIG. 41), an upper and a lower target are pre-set viacontroller 900 for the value of the OCP on the SE 817. The feedbackactivates current driven pH change when the potential is below the lowerbound and switch 802 cuts the current when above the upper bound. Thedata shows the OCP rising until the upper target is reached. Thefeedback then switches off the WE 816, which leads to a drop in the OCPas the buffer from the bulk starts to restore the local pH of solution700. When the OCP reaches the lower target, the current is re-initiatedusing switch 802 to increase the OCP again. This feedback mechanismallows a defined pH for solution 700 to be maintained close to the WE816. In another setup (shown in FIG. 42), the feedback can be activatedto continuously change the OCP so that a positive current is applied toactively increase the OCP until the upper bound is reached and then anegative current in applied in reverse to actively decrease the OCPuntil a lower bound is reached. This way, the system does not rely onpassive diffusion but actively monitors and adjusts the pH by electroniccontrol.

Further, FIG. 39 shows experimental results of the open circuitpotential voltage measured on a sense electrode adjacent to a workingelectrode with applied current. The working electrode is in a closedloop feedback with the sense electrode. The open circuit potential (OCP)of the working electrode as detected by the sense electrode is analogousto the pH of the solution near the working electrode. The feedback hasbeen set to apply current to the WE only when the sense electrode opencircuit potential is below 0.2V. The feedback is then bound between0.19V and 0.2V (target OCP), switching the potential on the workingelectrode ON below 0.19 and OFF above 0.2V. The shift at time 560 s is adisruption caused by pipetting the bulk solution into the workingelectrode. This perturbs the pH gradient that was generated by thecurrent applied to the working electrode causing the pH of the solutionto immediately return to the pH of the bulk solution. After pipetting isstopped, the closed loop feedback system is able to restore and thenmaintain the target OCP.

FIGS. 41-56 show schematics for a closed loop feedback setup, where theOCP between the sense electrode (SE) 816 and reference electrode (RE)822 is provided as input to a controller 900 that regulates the currentsource(s) 800-801 or potential source(s) 804-805 through one or moreswitches 802-803, 806-810. In this way, the system can be triggered onand off to increase or decrease the electrochemicalgeneration/consumption of H⁺ ions to balance the increase or decreasewith the rate of diffusion of buffering ions from bulk solution toachieve a specific pH value as defined by a target OCP (V_(TARGET))between the SE 816 and RE 822.

Example 20—Use of a pH Sensitive Coating

To further improve the closed loop pH control method, improved pHsensitivity can be incorporated by the addition of a pH sensitivecoating 840 on the working and/or sense electrode 815, 816, 817. Anexample of such a coating is PANI, which has been shown to haveexceptional pH sensitivity. PANI contains charged groups that interactwith the hydrogen ions and change the conductivity of the polymer. Theresponse of PANI as a function of pH is close to the Nernstian limit ofpH detection (59 mV/pH). The change in open circuit potential of PANI ishighly selective for H⁺ ions, unlike just a bare electrode surface thatis sensitive to other ions in solution other than just H⁺. FIG. 37 showsa response of the open circuit potential (OCP) as a function of pH wherethe surface of the electrode is coated with a pH sensitive PANI coating.The response of the OCP as a function of pH is shown by the slope of theline to be approximately 60 mV/pH which is close to the Nernstian limit.

Example 21—Device Designs

FIG. 40 shows example schematics of device designs utilizing the openloop method. In part A, a controlled current source is used while inpart B a controlled voltage source is used.

Various designs can be used in conjunction with the closed loop method.FIGS. 41, 43, 45, 47, 49, 51, and 53 show various designs for the closedloop method with a single controlled current source 800, and FIGS. 42,44, 46, 48, 50, 52, and 54-56 show alternative designs using a dualcontrolled current source 800, 801. The closed loop method can also beimplemented on designs with a PANI coated 840 sense electrode 817 (FIGS.43, 44, 47-50, and 53-56). The WE and SE may also be combined into asingle electrode 815 that is able to function as both the sense andworking electrodes (FIGS. 45-48). Various switches 802-803, 806-810 areused to connect and disconnect the current 800-801 and voltage sources804-805. FIGS. 41, 43, 45, and 47 shows a simple switch 802 forconnecting and disconnecting the current source 800. FIG. 49 shows asimilar switch 806 for connecting and disconnecting the voltage source804. FIGS. 42, 44, 46, and 48 shows a changeover switch 803 forswitching between the positive current source 800 and negative currentsource 801. FIG. 51 shows a similar changeover switch for switchingbetween positive voltage source 804 and negative voltage source 805. InFIGS. 51-54, switches 808-810 operate in conjunction with clock phasesPhi1 and Phi2. Switches 808 and 810 operate in conjunction with clockphase Phi1 and switch 809 in conjunction with Phi2. In each of the aboveexamples the various switches which are controlled by the controller 900based on feedback to allow for additional levels of control over the WEpotential input and SE measurement output.

FIG. 55 schematically shows the system architecture with a closed loopcontroller. This system controls the pH with only one target valueV_(TARGET) 907. The same architecture can be applied to electrochemicalcells with systems which use electric voltages instead of electriccurrents to modulate pH. In an example embodiment, the controller worksas follows. The input voltage (V_(in)) 830 is sampled 901 and compared902 to a target voltage V_(TARGET) 907 and the difference is amplified903 and processed by a loop filter 904. This could also be part of theloop filter and also be realized in different ways, e.g., as a switchedcapacitor amplifiers or a switched-capacitor loop filter. One examplefor a loop filter 904 is a PID-controller with a proportional part (P),an integrating part (I) and a differentiating part (D). The outputsignal of the loop filter 904 is compared with a fixed threshold by acomparator 905. The output of the comparator 905 can be positive ornegative. This is equivalent to a digital representation. This digitalsignal is then stored in a clocked module 906 like a flip-flop or alatch. In an example embodiment, the frequency of the clock 908 is muchhigher than the frequency which is determined by the inverse of thediffusion time constant of H⁺ ions. The output signal of the flip-flopthen determines if a positive or negative unity current is applied tothe WE 816. This scheme can also be applied to a system with only onecurrent source 800 or to a system with voltage sources 804, 805 insteadof current sources 800, 801. The system works as an “electrochemicaldelta-sigma-modulator,” where the quantization error of the unitycurrent sources is “shaped” by the transfer functions of theelectrochemical cell and the transfer function of the electronic loopfilter 904 and is distributed over a wide frequency spectrum determinedby the frequency of the clock CLK 908. The one-bit-output of thecomparator can be filtered by a digital filter 909, as shown in FIG. 55.This results in a digital representation of what the system has to applyto electrochemical cell in order to keep the pH at the target value.

There is flexibility in the controller architecture design in that ananalog controller, a digital controller, or a controller using bothanalog and digital signal processing can be used. FIG. 56 shows a secondcontroller architecture where the main difference from that of FIG. 55being the way how the target value for pH is set. In FIG. 55, the targetpH is set by an analog voltage V_(TARGET) 907. This V_(TARGET) 907 couldbe generated by a digital-to-analog converter (DAC). In FIG. 56, theinput voltage V_(in) 830 is digitized by an analog-to-digital converter(ADC) 911 and then compared 912 with a digital target value 910. Asnoted above, the electronics is clocked at a frequency 908 much higherthan the inverse of the diffusion time constant of the H⁺ ions.

The controllers in FIG. 56 can also be used to “measure” using theclosed loop method the electric current or voltage that is needed to seta certain target pH. This information can be used to characterize thesystem and derive stimuli for an open loop system. One example wherethis might be very useful is an array-structure of many sites where onesite is used to “measure” the correct values using the closed loop, andthose values are then applied according to the open-loop to many other“mirrored” sites.

FIG. 57 shows an illustration of example electrode configurations(routing not shown) for pH sensing. An illustration of the geometry ofthe working electrode and sense electrode includes: (A) sense electrodelocated adjacent to the working electrode, (B) sense electrodes locatedwithin the working electrode, (C) multiple sense electrodes for a singleworking electrode, and (D) interdigitated working and sense electrodes.The incorporation of the PANI surface is advantageous for improving theaccuracy of closed loop pH control. There are many different methods toincorporate a sense electrode for monitoring pH. For example, in stillanother alternative, the working electrode and sense electrode can alsobe one and the same by switching between active and passive/measuringsteps.

What is claimed is:
 1. A method comprising: a. adding anelectrochemically active agent which is a quinone derivative onto asupport of a biosensor; and b. adding an analyte that, in dependenceupon a change of a redox state of the quinone derivative, interacts witha probe on the support, wherein an electrode of the support isconfigured to cause the change in the redox state of the quinonederivative.
 2. The method of claim 1, wherein the change of the redoxstate of the quinone derivative allows the interaction between theanalyte and the probe to occur.
 3. The method of claim 1, wherein thechange of the redox state of the quinone derivative increases a bindingaffinity between the analyte and the probe.
 4. The method of claim 1,wherein the change of the redox state of the quinone derivativedecreases a binding affinity between the analyte and the probe.
 5. Amethod comprising: a. providing a biosensor comprising a support in anaqueous solution, wherein: i. the support comprising one or moreelectrodes, and a biomolecule interface layer having one or moreimmobilized probes thereon; and ii. the aqueous solution comprising aquinone derivative; b. adding a biomolecule analyte to the aqueoussolution; c. reacting the quinone derivative using the one or moreelectrodes to produce an amount of H⁺ ions and/or an amount of OH⁻ ions,wherein the pH of the aqueous solution close to the one or moreelectrodes is controlled by the amount of H⁺ ions and/or the amount ofOH⁻ ions produced; and d. collecting signals from the biosensor.
 6. Themethod of claim 5, wherein the one or more immobilized probes comprisesa pH sensitive probe.
 7. The method of claim 6, wherein the pH sensitiveprobe is a fluorescent protein and optionally a green fluorescentprotein.
 8. The method of claim 6, wherein a first area of thebiomolecule interface covers at least one area of the support notcovered by the one or more electrode and a second area of thebiomolecule interface covers at least one area of the one or moreelectrodes.
 9. The method of claim 8, wherein the pH sensitive probe isimmobilized on the first area and second area of the biomoleculeinterface.
 10. The method of claim 9, further comprising determining thepH of the solution near the one or more electrode using a fluorescenceintensity of an immobilized pH sensitive fluorescent protein immobilizedon the second area of the biomolecule interface layer.
 11. The method ofclaim 9, further comprising normalizing the fluorescence intensity ofthe immobilized pH sensitive fluorescent protein immobilized on thesecond area of the biomolecule interface layer with respect to thefluorescence intensity of the immobilized pH sensitive fluorescentprotein immobilized on the first area of the biomolecule interfacelayer, and the pH is determined using the normalized fluorescenceintensity.
 12. The method of claim 5, wherein the one or moreimmobilized probes comprises an immobilized enzyme and the biomoleculeanalyte is an enzyme substrate.
 13. The method of claim 5, wherein theone or more immobilized probes comprises an immobilized enzyme substrateand the biomolecule analyte is an enzyme.
 14. The method of claim 5,further comprising measuring the pH using a sense electrode wherein theone or more electrodes comprises the sense electrode.
 15. The method ofclaim 5, wherein the biosensor comprises a multisite array of test siteswith each test site having the support in the solution and wherein oneor more test conditions for each test site can be independently varied.16. The method of claim 15, wherein the pH of the solution close to theone or more electrodes in each test site is independently controlled.17. The method of claim 15, wherein collecting signals from thebiosensor include collecting varied signals from the multisite array oftest sites to obtain a collection of varied signals.
 18. A biosensorsystem comprising: a. a support that includes an electrode; b. amaterial on the support; c. an electrochemically active agent in anaqueous solution, wherein the electrode is configured to control achange of a redox state of the electrochemically active agent, and thematerial provides an indication of the change of the redox state.
 19. Abiosensor system comprising: a. a support that includes an electrode; b.a probe on the support; c. an electrochemically active agent which is aquinone derivative; and d. an analyte that interacts with the probe onthe support in dependence upon a change of a redox state of the quinonederivative, wherein the electrode is configured to control the change ofthe redox state of the quinone derivative.
 20. The biosensor of claim19, wherein the change of the redox state of the quinone derivativeallows the interaction between the analyte and the probe to occur. 21.The biosensor of claim 19, wherein the change of the redox state of thequinone derivative increases a binding affinity between the analyte andthe probe.
 22. The biosensor of claim 19, wherein the change of theredox state of the quinone derivative decreases a binding affinitybetween the analyte and the probe.
 23. The biosensor of claim 19,wherein: the support includes a biomolecule interface layer; a firstarea of the biomolecule interface layer covers at least one area of thesupport not covered by the electrode; and a second area of thebiomolecule interface layer covers at least one area of the electrode.24. A biosensor comprising a support in a solution, wherein: a. thesolution comprises a quinone derivative; b. the support comprises one ormore electrodes and a biomolecule interface layer having one or moreimmobilized probes thereon; and c. the biosensor is configured to modifythe pH of the solution near the one or more electrodes byelectrochemically oxidizing or reducing the quinone derivative toproduce H⁺ ions or OH⁻ ions.
 25. The biosensor of claim 24, wherein thesolution is an aqueous solution.
 26. The biosensor of claim 24, whereinthe one or more immobilized probes comprises a pH sensitive probe. 27.The biosensor of claim 26, wherein the pH sensitive probe is afluorescent protein and optionally a green fluorescent protein.
 28. Thebiosensor of claim 26, wherein a first area of the biomolecule interfacecovers at least one area of the support not covered by the one or moreelectrode and a second area of the biomolecule interface covers at leastone area of the one or more electrodes.
 29. The biosensor of claim 28,wherein the pH sensitive probe is immobilized on the first area andsecond area of the biomolecule interface.
 30. The biosensor of claim 29,wherein the biosensor is configured to normalize the fluorescenceintensity of the immobilized pH sensitive fluorescent proteinimmobilized on the second area of the biomolecule interface layer withrespect to the fluorescence intensity of the immobilized pH sensitivefluorescent protein immobilized on the first area of the biomoleculeinterface layer, and the biosensor is configured to determine the pHusing the normalized fluorescence intensity.
 31. The biosensor of claim29, wherein the biosensor is configured to determine the pH of thesolution near the one or more electrode using a fluorescence intensityof an immobilized pH sensitive fluorescent protein immobilized on thesecond area of the biomolecule interface layer.
 32. The biosensor ofclaim 24, wherein the one or more immobilized probes comprises animmobilized enzyme and the biomolecule analyte is an enzyme substrate.33. The biosensor of claim 24, wherein the one or more immobilizedprobes comprises an immobilized enzyme substrate and the biomoleculeanalyte is an enzyme.
 34. The biosensor of claim 24, wherein the one ormore electrodes comprises a sense electrode and the biosensor isconfigured to measure the pH using the sense electrode.
 35. Thebiosensor of claim 24, wherein the biosensor comprises a multisite arrayof test sites with each test site having the support in the solution andone or more test conditions for each test site can be independentlyvaried.
 36. The biosensor of claim 35, wherein the pH of the solutionclose to the one or more electrodes in each test site is independentlycontrolled.
 37. The biosensor of claim 35, wherein the biosensor isconfigured to obtain a collection of varied signals including variedsignals from the multisite array of test sites.